Galnac compositions for improving sirna bioavailability

ABSTRACT

Provided herein, are compositions comprising GalNAc moieties that may be conjugated to an oligonucleotide. The oligonucleotide may be a small interfering RNA or an antisense oligonucleotide. Also provided herein are methods of treatment that include administering the composition to a subject.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.63/320,431, filed Mar. 16, 2022, U.S. Provisional Application No.63/354,359, filed Jun. 22, 2022, and U.S. Provisional Application No.63/430,542, filed Dec. 6, 2022, which applications are incorporatedherein by reference.

INCORPORATION BY REFERENCE OF A SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled54462-735201_US.xml, created Mar. 13, 2023, which is 922,453 bytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

BACKGROUND

Cardiovascular, metabolic, and liver-related disorders are abundant, andmay affect a wide variety of persons. Improved therapeutics are neededfor treating these disorders.

SUMMARY

Disclosed herein is a compound represented by Formula (I) or (II):

-   -   or a salt thereof, wherein    -   J is an oligonucleotide;    -   each w is independently selected from any value from 1 to 20;    -   each v is independently selected from any value from 1 to 20;    -   n is selected from any value from 1 to 20;    -   m is selected from any value from 1 to 20;    -   z is selected from any value from 1 to 3, wherein        -   if z is 3, Y is C        -   if z is 2, Y is CR⁶, or        -   if z is 1, Y is C(R⁶)₂;    -   Q is selected from:        -   C₃₋₁₀ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl,            is optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            and —NH₂;    -   R¹ is a linker selected from:        -   —O—, —S—, —N(R⁷)—, —C(O)—, —C(O)N(R⁷)—, —N(R⁷)C(O)—,            —N(R⁷)C(O)N(R⁷)—, —OC(O)N(R⁷)—, —N(R⁷)C(O)O—, —C(O)O—,            —OC(O)—, —S(O)—, —S(O)₂—, —OS(O)₂—, —OP(O)(OR⁷)O—,            —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—,            —OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—,            —OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—, —OP(O)(N(R⁷)₂)NR⁷—,            —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and            —OPN(R⁷)₂NR⁷—;    -   each R² is independently selected from:        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —OR⁷,            —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   R³ and R⁴ are each independently selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁵ is independently selected from:        -   —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)R⁷, —C(O)OR⁷, and —C(O)N(R⁷)₂;    -   each R⁶ is independently selected from:        -   hydrogen;        -   halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; and        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁷ is independently selected from:        -   hydrogen;        -   C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,            —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to 10-membered            heterocycle; and        -   C₃₋₁₀ carbocycle, and 3- to 10-membered heterocycle, each of            which is optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl,            —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₁₋₆ alkyl, C₂₋₆, C₂₋₆            alkynyl, C₃₋₁₀ carbocycle, 3- to 10-membered heterocycle,            and C₁₋₆ haloalkyl.            In some embodiments, each w is independently selected from            any value from 1 to 10. In some embodiments, each w is            independently selected from any value from 1 to 5. In some            embodiments, each w is 1. In some embodiments, each v is            independently selected from any value from 1 to 10. In some            embodiments, each v is independently selected from any value            from 1 to 5. In some embodiments, each v is 1. In some            embodiments, n is selected from any value from 1 to 10. In            some embodiments, n is selected from any value from 1 to 5.            In some embodiments, n is 2. In some embodiments, m is            selected from any value from 1 to 10. In some embodiments, m            is selected from any value from 1 to 5. In some embodiments,            m is selected from 1 and 2. In some embodiments, z is 3 and            Y is C. In some embodiments, Q is selected from C₅            carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷. In some embodiments, Q is selected            from C₅₋₆ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, and —NH₂. In some embodiments, Q is selected from            phenyl and cyclohexyl, each of which is optionally            substituted with one or more substituents independently            selected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. In            some embodiments, Q is selected from phenyl. In some            embodiments, Q is selected from cyclohexyl.            In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—,            —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—,            —OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—,            —OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—, —OP(O)(N(R⁷)₂)NR⁷—,            —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and —OPN(R⁷)₂NR⁷.            In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—,            —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—,            —OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—,            —OP(O)(O⁻)S—, and —OP(OR⁷)O—. In some embodiments, R¹ is            selected from —OP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(O⁻)O—,            —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(OR⁷)O—. In some            embodiments. R¹ is selected from —OP(O)(OR⁷)O— and            —OP(OR⁷)O—.            In some embodiments, R² is selected from C₁₋₃ alkyl            substituted with one or more substituents independently            selected from halogen, —OR⁷, —OC(O)R⁷, —SR⁷, —N(R⁷)₂,            —C(O)R⁷, and —S(O)R⁷. In some embodiments, R² is selected            from C₁₋₃ alkyl substituted with one or more substituents            independently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and            —N(R⁷)₂. In some embodiments, R² is selected from C₁₋₃ alkyl            substituted with one or more substituents independently            selected from —OR⁷ and —OC(O)R⁷. In some embodiments, R³ is            selected from halogen, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —OC(O)R⁷, and —S(O)R⁷. In some embodiments, R³ is selected            from —OR⁷, —SR⁷, —OC(O)R⁷, and —N(R⁷)₂. In some embodiments,            R³ is selected from —OR⁷— and —OC(O)R⁷. In some embodiments,            R⁴ is selected from halogen, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —OC(O)R⁷, and —S(O)R⁷. In some embodiments, R⁴ is selected            from —OR⁷—SR⁷, —OC(O)R⁷, and —N(R⁷)₂. In some embodiments.            R⁴ is selected from —OR⁷— and —OC(O)R⁷. In some embodiments,            R⁵ is selected from —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, and —N(R⁷)C(O)OR⁷. In some embodiments, R⁵            is selected from —OC(O)R⁷ and —N(R⁷)C(O)R⁷. In some            embodiments, each R⁷ is independently selected from:            hydrogen; and C₁₋₄ alkyl optionally substituted with one or            more substituents independently selected from halogen, —CN,            —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₄ alkyl,            —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, or 3- to            10-membered heterocycle. In some embodiments, each R⁷ is            independently selected from C₁₋₆alkyl optionally substituted            with one or more substituents independently selected from            halogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl,            —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, and —NH(C₁₋₆ alkyl). In some            embodiments, each R⁷ is independently selected from C₁₋₆            alkyl optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, and —SH. In            some embodiments, w is 1; v is 1; n is 2; m is 1 or 2; z is            3 and Y is C; Q is phenyl or cyclohexyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, and C₁₋₃ alkyl; R¹ is selected from —OP(O)(OR⁷)O—,            —OP(S)(OR⁷)O—, —OP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and            —OP(OR⁷)O—; R² is C₁ alkyl substituted with —OH or            —OC(O)CH₃;            R³ is —OH or —OC(O)CH₃; R⁴ is —OH or —OC(O)CH₃; and R⁵ is            —NH(O)CH₃. In some embodiments, the compound comprises:

In some embodiments, the oligonucleotide (J) is attached at a 5′ end ora3′ end of the oligonucleotide. In some embodiments, the oligonucleotidecomprises DNA. In some embodiments, the oligonucleotide comprises RNA.In some embodiments, the oligonucleotide comprises one or more modifiedinternucleoside linkages. In some embodiments, the one or more modifiedinternucleoside linkages comprise alkylphosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,or carboxymethyl ester, or a combination thereof. In some embodiments,the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 modified internucleoside linkages. In someembodiments, the oligonucleotide comprises one or more modifiednucleosides. In some embodiments, the one or more modified nucleosidescomprise a locked nucleic acid (LNA), hexitol nucleic acid (HLA),cyclohexene nucleic acid (CeNA), 2′-methoxyethyl, 2′-O-alkyl,2′-O-allyl, 2′-O-allyl, 2′-fluoro, or 2′-deoxy, or a combinationthereof. In some embodiments, the one or more modified nucleosidescomprise a 2′,4′ constrained ethyl nucleoside, a 2′-O-methyl nucleoside,2′-deoxyfluoro nucleoside. 2′-O—N-methylacetamido (2′-O-NMA) nucleoside,a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleoside,2′-O-aminopropyl (2′-O-AP) nucleoside, 2′-ara-F, 2′fluoro, or 2′O-alkyl, or a combination thereof. In some embodiments, theoligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or more modified nucleosides. In someembodiments, the oligonucleotide comprises a lipid attached at a 3′ or5′ terminus of the oligonucleotide. In some embodiments, the lipidcomprises cholesterol, myristoyl, palmitoyl, stearoyl, lithocholoyl,docosanoyl, docosahexaenoyl, myristyl, palmityl stearyl, orα-tocopherol, or a combination thereof. In some embodiments, theoligonucleotide comprises an arginine-glycine-aspartic acid (RGD)peptide attached at a 3′ or 5′ terminus of the oligonucleotide. In someembodiments, the ROD peptide comprises Cyclo(-Arg-Gly-Asp-D-Phe-Cys),Cyclo(-Arg-Gly-Asp-D-Phe-Lys), Cyclo(-Arg-Gly-Asp-D-Phe-azido), an aminobenzoic acid derived RGD, or a combination thereof. In some embodiments,the oligonucleotide comprises a small interfering RNA (siRNA) comprisinga sense strand and an antisense strand. In some embodiments, the sensestrand is 12-30 nucleosides in length. In some embodiments, theantisense strand is 12-30 nucleosides in length. In some embodiments,the sense strand and the antisense strand form a double-stranded RNAduplex. In some embodiments, a first base pair of the double-strandedRNA duplex is an AU base pair. In some embodiments, the sense strand orthe antisense strand comprises a 3′ overhang. In some embodiments, the3′ overhang comprises 1, 2, or more nucleosides. In some embodiments,the sense strand comprises any one of modification patterns 1S to 6S, or1S #2 to 6S #2. In some embodiments, the antisense strand comprises anyone of modification patterns 1AS to 9AS. In some embodiments, theoligonucleotide comprises an antisense oligonucleotide (ASO). In someembodiments, the ASO is 12-30 nucleosides in length. In someembodiments, the ASO comprises modification pattern ASO1. In someembodiments, the compound binds to an asialoglycoprotein receptor. Insome embodiments, the compound targets a hepatocyte.

Disclosed herein is a pharmaceutical composition comprising the compoundof any one of the compounds described herein, and a pharmaceuticallyacceptable carrier, excipient, or diluent. In some embodiments, thepharmaceutical composition is sterile. In some embodiments, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier. In some embodiments, the pharmaceutically acceptable carriercomprises water, a buffer, or a saline solution. In some embodiments,the oligonucleotide targets a target mRNA and when administered to asubject in an effective amount decreases the target mRNA or a targetprotein by at least 10%.

Disclosed herein is a method of decreasing a target mRNA or targetprotein in a subject in need thereof, comprising administering aneffective amount of the pharmaceutical composition of any one of thecompounds described herein. In some embodiments, the effective amountdecreases a measurement of the target mRNA or target protein in thesubject, relative to a baseline target mRNA or target proteinmeasurement. In some embodiments, the effective amount treats a disorderin the subject. In some embodiments, the effective amount decreases ameasurement of a symptom or parameter related to the disorder in thesubject, relative to a baseline symptom or parameter measurement. Insome embodiments, the measurement of the symptom or the parameterrelated to the disorder in the subject is decreased for at least 10days. In some embodiments, the measurement of the symptom or theparameter related to the disorder in the subject is decreased for atleast 100 days. In some embodiments, the disorder comprises a metabolicdisorder. In some embodiments, the disorder comprises a liver disorder.

Disclosed herein is a compound represented by Formula (A) or (B):

-   -   or a salt thereof, wherein    -   each w is independently selected from any value from 1 to 20;    -   each v is independently selected from any value from 1 to 20;    -   n is selected from any value from 1 to 20;    -   m is selected from any value from 1 to 20;    -   z is selected from any value from 1 to 3, wherein        -   if z is 3, Y is C        -   if z is 2, Y is CR⁶, or        -   if z is 1, Y is C(R⁶)₂;    -   Q is selected from:        -   C₃₋₁₀ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl,            is optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            and —NH₂;    -   R¹ is selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, —S(O)₂R⁷, —OS(O)₂R⁷, —OP(O)(OR⁷)₂,            —OP(S)(OR⁷)₂, —SP(O)(OR⁷)₂, —OP(O)(SR⁷)(OR⁷),            —OP(O)(OR⁷)N(R⁷)₂, —OP(S)(OR⁷)N(R⁷)₂, —SP(O)(OR⁷)N(R⁷)₂,            —OP(O)(SR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(S)(N(R⁷)₂)₂,            —SP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂, —SP(OR⁷)₂, —OP(OR⁷)(SR⁷),            —OP(OR⁷)N(R⁷)₂, —OP(SR⁷)N(R⁷)₂, —SP(OR⁷)N(R⁷)₂,            —OP(N(R⁷)₂)₂, and —SP(N(R⁷)₂)₂:    -   each R² is independently selected from:        -   C₁₋₆alkyl optionally substituted with one or more            substituents independently selected from halogen, —OR⁷,            —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   R³ and R⁴ are each independently selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁵ is independently selected from:        -   —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)R⁷, —C(O)OR⁷, and —C(O)N(R⁷)₂;    -   each R⁶ is independently selected from:        -   hydrogen;        -   halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; and        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁷ is independently selected from:        -   hydrogen;        -   C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,            —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to 10-membered            heterocycle; and        -   C₃₋₁₀ carbocycle, and 3- to 10-membered heterocycle, each of            which is optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl,            —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₁₋₆ alkyl, C₂₋₆ alkenyl,            C₂₋₆ alkynyl, C₃₋₁₀ carbocycle, 3- to 10-membered            heterocycle, and C₁₋₆ haloalkyl.            In some embodiments, each w is independently selected from            any value from 1 to 10. In some embodiments, each w is            independently selected from any value from 1 to 5. In some            embodiments, each w is 1. In some embodiments, each v is            independently selected from any value from 1 to 10. In some            embodiments, each v is independently selected from any value            from 1 to 5. In some embodiments, each v is 1. In some            embodiments, n is selected from any value from 1 to 10. In            some embodiments, n is selected from any value from 1 to 5.            In some embodiments, n is 2. In some embodiments, m is            selected from any value from 1 to 10. In some embodiments, m            is selected from any value from 1 to 5. In some embodiments,            m is selected from 1 and 2. In some embodiments, z is 3 and            Y is C. In some embodiments, Q is selected from C₅₋₆            carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷. In some embodiments, Q is selected            from C₅₋₆ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, and —NH₂. In some embodiments, Q is selected from            phenyl and cyclohexyl, each of which is optionally            substituted with one or more substituents independently            selected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. In            some embodiments, Q is selected from phenyl. In some            embodiments, Q is selected from cyclohexyl.            In some embodiments, R¹ is selected from —OP(O)(OR⁷)₂,            —OP(O)(OR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂,            —OP(OR⁷)N(R⁷)₂, and —OP((NR⁷)₂)₂. In some embodiments, R¹ is            selected from —OP(O)(OR⁷)₂ and —OP(OR⁷)N(R⁷)₂. In some            embodiments, R¹ is selected from —OP(O)(OCH₂CH₃)OH and            —OP(OCH₂CH₂CN)N(CH(CH)₂)₂. In some embodiments, R¹ is            —OP(OCH₂CH₂CN)N(CH(CH₃)₂)₂. In some embodiments, R² is            selected from C₁₋₃ alkyl substituted with one or more            substituents independently selected from halogen, —OR⁷,            —OC(O)R⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, and —S(O)R⁷. In some            embodiments, R² is selected from C₁₋₃ alkyl substituted with            one or more substituents independently selected from —OR⁷,            —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In some embodiments, R² is            selected from C₁₋₃ alkyl substituted with one or more            substituents independently selected from —OR⁷ and —OC(O)R⁷.            In some embodiments, R³ is selected from halogen, —OR⁷,            —SR⁷, —N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some            embodiments. R³ is selected from —OR⁷—SR⁷, —OC(O)R⁷, and            —N(R⁷)₂. In some embodiments, R³ is selected from —OR⁷— and            —OC(O)R⁷. In some embodiments, R⁴ is selected from halogen,            —OR, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some            embodiments. R⁴ is selected from —OR⁷, —SR⁷, —OC(O)R⁷, and            —N(R⁷)₂. In some embodiments, R⁴ is selected from —OR⁷— and            —OC(O)R⁷. In some embodiments, R⁵ is selected from —OC(O)R⁷,            —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, and            —N(R⁷)C(O)OR⁷. In some embodiments, R⁵ is selected from            —OC(O)R⁷ and —N(R⁷)C(O)R⁷. In some embodiments, each R⁷ is            independently selected from: hydrogen; and C₁₋₆ alkyl            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,            —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, or 3- to 10-membered            heterocycle. In some embodiments, each R⁷ is independently            selected from C₁₋₆alkyl optionally substituted with one or            more substituents independently selected from halogen, —CN,            —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl,            —N(C₁₋₆ alkyl)₂, and —NH(C₁₋₆ alkyl). In some embodiments,            each R⁷ is independently selected from C₁₋₆ alkyl optionally            substituted with one or more substituents independently            selected from halogen, —CN, —OH, and —SH. In some            embodiments, w is 1; v is 1; n is 2; m is 1 or 2; z is 3 and            Y is C; Q is phenyl or cyclohexyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, and C₁₋₃ alkyl; R¹ is selected from —OP(O)(OR⁷)₂ and            —OP(OR⁷)N(R⁷) R² is C₁ alkyl substituted with —OH or            —OC(O)CH₃; R³ is —OH or —OC(O)CH₃; R⁴ is —OH or —OC(O)CH₃;            and R⁵ is —NH(O)CH₃. In some embodiments, the compound            comprises:

DETAILED DESCRIPTION

N-Acetylgalactosamine (GalNAc), is an amino sugar derivative ofgalactose. GalNAc and GalNAc-containing moieties may bind lectins suchas asialoglycoprotein receptors. These receptors may be included onhepatocytes. Thus, GalNAc may target an oligonucleotide to a hepatocyteor to the liver.

Provided herein, are GalNAc moieties. These GalNAc moieties may beconjugated to an oligonucleotide such as a small interfering RNA (siRNA)or antisense oligonucleotide (ASO). The oligonucleotide conjugated tothe GalNAc moiety may be administered to a subject, targeted to a liveror hepatocyte, or used to treat a liver related disorder in the subject.

I. COMPOSITIONS

Provided herein, in some embodiments, are compositions comprising anoligonucleotide and an N-Acetylgalactosamine (GalNAc) moiety. In someembodiments, the composition comprises an oligonucleotide. Theoligonucleotide may inhibit a target gene or oligonucleotide. Theoligonucleotide may bind a target oligonucleotide. In some embodiments,the composition is used in a method described herein.

Provided herein, in some embodiments, are compounds comprising anoligonucleotide and a GalNAc moiety. In some embodiments, the compoundcomprises an oligonucleotide. The oligonucleotide may bind to a targetoligonucleotide. In some embodiments, the compound is used in a methoddescribed herein. In some embodiments, the compound is included in acomposition described herein.

The oligonucleotide of the compound or composition described herein maycomprise a small interfering RNA (siRNA) or an antisense oligonucleotide(ASO).

Some embodiments include a composition comprising a GalNAc moiety, andan oligonucleotide that when administered to a subject in an effectiveamount decreases a target mRNA or protein level in a cell, fluid ortissue of the subject. Some embodiments include a composition comprisinga GalNAc moiety, and an oligonucleotide that when administered to asubject in an effective amount decreases a target mRNA or protein levelin liver tissue or in a hepatocyte. In some embodiments, the compositioncomprises a GalNAc moiety, and an oligonucleotide that when administeredto a subject in an effective amount decreases levels of a target e.g.mRNA in a cell or tissue. In some embodiments, the cell is a hepatocyte.In some embodiments, the tissue is liver tissue. Some embodimentsinclude a composition comprising a GalNAc moiety, and an oligonucleotidethat when administered to a subject in an effective amount decreases atarget mRNA level in liver tissue. Some embodiments include acomposition comprising a GalNAc moiety, and an oligonucleotide that whenadministered to a subject in an effective amount decreases a target mRNAlevel in a hepatocyte.

In some embodiments, the decrease in the target oligonucleotide level isspecific to a hepatocyte in relation to another cell type. In someembodiments, the decrease in the target RNA level is specific to ahepatocyte in relation to another cell type. In some embodiments, thedecrease in the target mRNA level is specific to a hepatocyte inrelation to another cell type. In some embodiments, the decrease in thetarget protein level is specific to a hepatocyte in relation to anothercell type. In some embodiments, the decrease in the targetoligonucleotide level is specific to liver tissue in relation to anothertissue type. In some embodiments, the decrease in the target RNA levelis specific to liver in relation to another tissue type. In someembodiments, the decrease in the target mRNA level is specific to liverin relation to another tissue type. In some embodiments, the decrease inthe target protein level is specific to liver in relation to anothertissue type.

In some embodiments, the composition comprises a GalNAc moiety, and anoligonucleotide that binds to a target oligonucleotide, which whenadministered to a subject in an effective amount decreases the targetoligonucleotide levels in a cell or tissue. In some embodiments, thetarget oligonucleotide levels are decreased by about 2.5% or more, about5% or more, or about 7.5% or more, as compared to prior toadministration. In some embodiments, the target oligonucleotide levelsare decreased by about 10% or more, as compared to prior toadministration. In some embodiments, the target oligonucleotide levelsare decreased by about 20% or more, about 30% or more, about 40% ormore, about 50% or more, about 60% or more, about 70% or more, about 80%or more, about 90% or more, or about 100%, as compared to prior toadministration. In some embodiments, the target oligonucleotide levelsare decreased by no more than about 2.5%, no more than about 5%, or nomore than about 7.5%, as compared to prior to administration. In someembodiments, the target oligonucleotide levels are decreased by no morethan about 10%, as compared to prior to administration. In someembodiments, the target oligonucleotide levels are decreased by no morethan about 20%, no more than about 30%, no more than about 40%, no morethan about 50%, no more than about 60%, no more than about 70%, no morethan about 80%, no more than about 90%, or no more than about 100%, ascompared to prior to administration. In some embodiments, the targetoligonucleotide levels are decreased by 2.5%, 5%, 7.5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%, or by a range defined by any of the two aforementionedpercentages.

In some embodiments, the composition comprises a GalNAc moiety, and anoligonucleotide that binds to a target mRNA, which when administered toa subject in an effective amount decreases the target mRNA levels in acell or tissue. In some embodiments, the target mRNA levels aredecreased by about 2.5% or more, about 5% or more, or about 7.5% ormore, as compared to prior to administration. In some embodiments, thetarget mRNA levels are decreased by about 10% or more, as compared toprior to administration. In some embodiments, the target mRNA levels aredecreased by about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 70% or more, about 80% ormore, about 90% or more, or about 100%, as compared to prior toadministration. In some embodiments, the target mRNA levels aredecreased by no more than about 2.5%, no more than about 5%, or no morethan about 7.5%, as compared to prior to administration. In someembodiments, the target mRNA levels are decreased by no more than about10%, as compared to prior to administration. In some embodiments, thetarget mRNA levels are decreased by no more than about 20%, no more thanabout 30%, no more than about 40%, no more than about 50%, no more thanabout 60%, no more than about 70%, no more than about 80%, no more thanabout 90%, or no more than about 100%, as compared to prior toadministration. In some embodiments, the target mRNA levels aredecreased by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or by a rangedefined by any of the two aforementioned percentages.

In some embodiments, the composition comprises a GalNAc moiety, and anoligonucleotide that binds to an oligonucleotide encoding a targetprotein, which when the composition is administered to a subject in aneffective amount decreases the target protein levels in a cell ortissue. In some embodiments, the cell is a hepatocyte. In someembodiments, the tissue is liver tissue. In some embodiments, the targetprotein levels are decreased by about 2.5% or more, about 5% or more, orabout 7.5% or more, as compared to prior to administration. In someembodiments, the target protein levels are decreased by about 10% ormore, as compared to prior to administration. In some embodiments, thetarget protein levels are decreased by about 20% or more, about 30% ormore, about 40% or more, about 50% or more, about 60% or more, about 70%or more, about 80% or more, about 90% or more, or about 100%, ascompared to prior to administration. In some embodiments, the targetprotein levels are decreased by no more than about 2.5%, no more thanabout 5%, or no more than about 7.5%, as compared to prior toadministration. In some embodiments, the target protein levels aredecreased by no more than about 10%, as compared to prior toadministration. In some embodiments, the target protein levels aredecreased by no more than about 20%, no more than about 30%, no morethan about 40%, no more than about 50%, no more than about 60%, no morethan about 70%, no more than about 80%, no more than about 90%, or nomore than about 100%, as compared to prior to administration. In someembodiments, the target protein levels are decreased by 2.5%, 5%, 7.5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%, or by a range defined by any of the twoaforementioned percentages.

In some embodiments, the composition comprises a GalNAc moiety, and anoligonucleotide that binds to a target oligonucleotide (e.g. mRNA) andwhen administered to a subject in an effective amount decreases anadverse phenotype (e.g. a symptom of a disorder associated with thetarget oligonucleotide). In some embodiments, the adverse phenotype isdecreased by about 2.5% or more, about 5% or more, or about 7.5% ormore, as compared to prior to administration. In some embodiments, theadverse phenotype is decreased by about 10% or more, as compared toprior to administration. In some embodiments, the adverse phenotype isdecreased by about 20% or more, about 30% or more, about 40% or more,about 50% or more, about 60% or more, about 70% or more, about 80% ormore, about 90% or more, or about 100%, as compared to prior toadministration. In some embodiments, the adverse phenotype is decreasedby no more than about 2.5%, no more than about 5%, or no more than about7.5%, as compared to prior to administration. In some embodiments, theadverse phenotype is decreased by no more than about 10%, as compared toprior to administration. In some embodiments, the adverse phenotype isdecreased by no more than about 20%, no more than about 30%, no morethan about 40%, no more than about 50%, no more than about 60%, no morethan about 70%, no more than about 80%, no more than about 90%, or nomore than about 100%, as compared to prior to administration. In someembodiments, the adverse phenotype is decreased by 2.5%, 5%/l, 7.5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%, or by a range defined by any of the twoaforementioned percentages.

In some embodiments, the composition comprises a GalNAc moiety, and anoligonucleotide that binds to a target oligonucleotide (e.g. mRNA) andwhen administered to a subject in an effective amount increases aprotective phenotype (e.g. protective against a disorder). In someembodiments, the protective phenotype is increased by about 2.5% ormore, about 5% or more, or about 7.5% or more, as compared to prior toadministration. In some embodiments, the protective phenotype isincreased by about 10% or more, as compared to prior to administration.In some embodiments, the protective phenotype is increased by about 20%or more, about 30% or more, about 40% or more, about 50% or more, about60% or more, about 70% or more, about 80% or more, about 90% or more, orabout 100% or more, as compared to prior to administration. In someembodiments, the protective phenotype is increased by about 200% ormore, about 300% or more, about 400% or more, about 500% or more, about600% or more, about 700% or more, about 800% or more, about 900% ormore, or about 1000% or more, as compared to prior to administration. Insome embodiments, the protective phenotype is increased by no more thanabout 2.5%, no more than about 5%, or no more than about 7.5%, ascompared to prior to administration. In some embodiments, the protectivephenotype is increased by no more than about 10%, as compared to priorto administration. In some embodiments, the protective phenotype isincreased by no more than about 20%, no more than about 30%, no morethan about 40%, no more than about 50%, no more than about 60%, no morethan about 70%, no more than about 80%, no more than about 90%, or nomore than about 100%, as compared to prior to administration. In someembodiments, the protective phenotype is increased by no more than about200%, no more than about 300%, no more than about 400%, no more thanabout 500%, no more than about 600%, no more than about 700%, no morethan about 800%, no more than about 900%, or no more than about 1000%,as compared to prior to administration. In some embodiments, theprotective phenotype is increased by 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or1000%, or by a range defined by any of the two aforementionedpercentages.

A. GalNAc Moieties and Compounds

Provided herein, in some embodiments, are compositions comprising aGalNAc moiety. Provided herein, in some embodiments, are compositionscomprising a GalNAc moiety and an oligonucleotide. In some embodiments,a composition comprising GalNAc moiety and an oligonucleotide isdescribed by a compound of Formula (I) or Formula (II). In someembodiments the oligonucleotide of Formula (I) or Formula (II), is J. Insome embodiments the GalNAc moiety of Formula (I) or Formula (II) is themolecular moiety bound to J. The oligonucleotide may comprise a smallinterfering RNA (siRNA) or an antisense oligonucleotide (ASO).

Provided herein, in some embodiments, is a compound represented byFormula (I) or (II):

-   -   or a salt thereof, wherein    -   J is an oligonucleotide;    -   each w is independently selected from any value from 1 to 20;    -   each v is independently selected from any value from 1 to 20;    -   n is selected from any value from 1 to 20;    -   m is selected from any value from 1 to 20;    -   z is selected from any value from 1 to 3, wherein        -   if z is 3, Y is C        -   if z is 2, Y is CR⁶, or        -   if z is 1, Y is C(R⁶)₂;    -   Q is selected from:        -   C₃₋₁₀ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl,            is optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            and —NH₂;    -   R¹ is a linker selected from:        -   —O—, —S—, —N(R⁷)—, —C(O)—, —C(O)N(R⁷)—, —N(R⁷)C(O)—,            —N(R⁷)C(O)N(R⁷)—, —OC(O)N(R⁷)—, —N(R⁷)C(O)O—, —C(O)O—,            —OC(O)—, —S(O)—, —S(O)₂—, —OS(O)₂—, —OP(O)(OR⁷)O—,            —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—,            —OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—,            —OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—, —OP(O)(N(R⁷)₂)NR⁷—,            —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and            —OPN(R⁷)₂NR⁷—;    -   each R² is independently selected from:        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —OR⁷,            —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   R³ and R⁴ are each independently selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁵ is independently selected from:        -   —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)R⁷, —C(O)OR⁷, and —C(O)N(R⁷)₂;    -   each R⁶ is independently selected from:        -   hydrogen;        -   halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; and        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁷ is independently selected from:        -   hydrogen;        -   C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₄ alkyl, —N(C₁₋₆ alkyl)₂,            —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to 10-membered            heterocycle; and        -   C₃₋₁₀ carbocycle, and 3- to 10-membered heterocycle, each of            which is optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl,            —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₁₋₆alkyl, C₂₋₆ alkenyl,            C₂₋₄ alkynyl, C₃₋₁₀ carbocycle, 3- to 10-membered            heterocycle, and C₁₋₆ haloalkyl.

In some embodiments, each w is independently selected from any valuefrom 1 to 20. In some embodiments, each w is independently selected fromany value from 1 to 15. In some embodiments, each w is independentlyselected from any value from 1 to 10. In some embodiments, each w isindependently selected from any value from 1 to 5. In some embodiments,each w is independently selected from any value from 1 to 4. In someembodiments, each w is independently selected from any value from 1 to3. In some embodiments, each w is independently selected from any valuefrom 1 to 2. In some embodiments, each w is independently 1. In someembodiments, w is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

In some embodiments, each v is independently selected from any valuefrom 1 to 20. In some embodiments, each v is independently selected fromany value from 1 to 15. In some embodiments, each v is independentlyselected from any value from 1 to 10. In some embodiments, each v isindependently selected from any value from 1 to 5. In some embodiments,each v is independently selected from any value from 1 to 4. In someembodiments, each v is independently selected from any value from 1 to3. In some embodiments, each v is independently selected from any valuefrom 1 to 2. In some embodiments, each v is independently 1. In someembodiments, v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

In some embodiments, n is selected from any value from 1 to 20. In someembodiments, n is selected from any value from 1 to 15. In someembodiments, n is selected from any value from 1 to 10. In someembodiments, n is selected from any value from 1 to 9. In someembodiments, n is selected from any value from 1 to 8. In someembodiments, n is selected from any value from 1 to 7. In someembodiments, n is selected from any value from 1 to 6. In someembodiments, n is selected from any value from 1 to 5. In someembodiments, n is selected from any value from 1 to 4. In someembodiments, n is selected from any value from 2 to 4. In someembodiments, n is selected from any value from 1 to 3. In someembodiments, n is 2 or 3. In some embodiments, n is 3. In someembodiments, n is selected from any value from 1 to 2. In someembodiments, n is 2. In some embodiments, n is 1. In some embodiments, nis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20.

In some embodiments, m is selected from any value from 1 to 20. In someembodiments, m is selected from any value from 1 to 15. In someembodiments, m is selected from any value from 1 to 10. In someembodiments, m is selected from any value from 1 to 9. In someembodiments, m is selected from any value from 1 to 8. In someembodiments, m is selected from any value from 1 to 7. In someembodiments, m is selected from any value from 3 to 7. In someembodiments, m is selected from any value from 1 to 6. In someembodiments, m is selected from any value from 2 to 6. In someembodiments, m is selected from any value from 3 to 6. In someembodiments, m is selected from any value from 4 to 6. In someembodiments, m is 6. In some embodiments, m is selected from any valuefrom 1 to 5. In some embodiments, m is selected from any value from 3 to5. In some embodiments, m is 5. In some embodiments, m is 4 or 5. Insome embodiments, m is selected from any value from 1 to 4. In someembodiments, m is 4. In some embodiments, m is 3 or 4. In someembodiments, m is selected from any value from 2 to 4. In someembodiments, m is selected from any value from 1 to 3. In someembodiments, m is 3. In some embodiments, m is selected from any valuefrom 1 to 2. In some embodiments, m is 2. In some embodiments, m is 1.In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

In some embodiments, z is selected from any value from 1 to 3. In someembodiments, z is 3 and Y is C. In some embodiments, z is 2 and Y isCR⁶. In some embodiments, z is 1 and Y is C(R⁶)₂.

In some embodiments, Q is selected from C₃₋₁₀ carbocycle optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,—OC(O)R⁷, —S(O)R, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl, is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, and —NH₂. In some embodiments, Q isselected from C₃₋₆ carbocycle optionally substituted with one or moresubstituents independently selected from halogen, —CN, —NO₂, —OR⁷, —SR⁷,—N(R)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, —S(O)R⁷, and C₁₋₆alkyl, wherein the C₁₋₆ alkyl, is optionally substituted with one ormore substituents independently selected from halogen, —CN, —OH, —SH,—NO₂, and —NH₂. In some embodiments, Q is selected from C₁₋₆ carbocycleoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, Qis selected from C₆ carbocycle optionally substituted with one or moresubstituents independently selected from halogen, —CN, —NO₂, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, Q is selected from C₅ carbocycle optionally substitutedwith one or more substituents independently selected from halogen, —CN,—NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and—S(O)R⁷. In some embodiments, Q is selected from C₅₋₆ carbocycleoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. In someembodiments, Q is selected from phenyl, cyclohexyl, cyclopentadiene, andcyclopentyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,and —NH₂. In some embodiments, Q is selected from phenyl and cyclohexyl,each of which is optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. Insome embodiments, Q is selected from phenyl and cyclohexyl. In someembodiments, Q is phenyl. In some embodiments, Q is cyclohexyl.

In some embodiments, R¹ is a linker selected from —O—, —S—, —N(R⁷)—,—C(O)—, —C(O)N(R⁷)—, —N(R⁷)C(O)—, —N(R⁷)C(O)N(R⁷)—, —OC(O)N(R⁷)—,—N(R⁷)C(O)O—, —C(O)O—, —OC(O)—, —S(O)—, —S(O)₂—, —OS(O)₂—,—OP(O)(OR⁷)O—, —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—,—OP(O)(OR⁷)S—, —OP(O)(OR⁷)NR⁷—, —OP(O)(N(R⁷)₂)NR⁷—, —OP(OR⁷)O—,—OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and —OPN(R⁷)₂NR⁷—. In some embodiments,R¹ is a linker selected from —O—, —S—, —N(R⁷)—, —C(O)—, —C(O)N(R⁷)—,—N(R⁷)C(O)—, —N(R⁷)C(O)N(R⁷)—, —OC(O)N(R⁷)—, —N(R⁷)C(O)O—, —C(O)O—,—OC(O)—, —S(O)—, —S(O)₂—, —OS(O)₂—, —OP(O)(OR⁷)O—, —SP(O)(OR⁷)O—,—OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—, —OP(O)(O⁻)O—, —SP(O)(O)O—,—OP(S)(O⁻)O—, —OP(O)(S⁻)O—, —OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—,—OP(O)(N(R⁷)₂)NR⁷—, —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and—OPN(R⁷)₂NR⁷—. In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—,—SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—, —OP(O)(O)O—,—SP(O)(O)O—, —OP(S)(O⁻)O—, —OP(O)(S)O—, —OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—,—OP(O)(N(R⁷)₂)NR⁷—, —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and—OPN(R⁷)₂NR⁷. In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—,—SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(O)(OR⁷)S—,—OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S)O—, —OP(O)(O⁻)S—, and—OP(OR⁷)O—. In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—,—OP(S)(OR⁷)O—, —OP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(OR⁷)O—.In some embodiments, R¹ is selected from —OP(O)(OR⁷)O— and —OP(OR⁷)O—.In some embodiments, R¹ is a linker selected from —OP(O)(OH)O—,—SP(O)(OH)O—, —OP(S)(OH)O—, —OP(O)(SH)O—, —OP(O)(OH)S—, —OP(O)(O⁻)O—,—SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(O)(O⁻)S—. In someembodiments, R¹ is a selected from —OP(O)(OR⁷)O—, —OP(O)(OR⁷)NR⁷—,—OP(O)(N(R⁷)₂)NR⁷—, —OP(OR⁷)O—, —OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and—OPN(R⁷)₂NR⁷—. In some embodiments, R¹ is selected from —S—, —S(O)—,—S(O)₂—, —OS(O)₂, —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—,—OP(O)(OR⁷)S—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and—OP(O)(O⁻)S—. In some embodiments, R¹ is selected from —S—, —S(O)—,—S(O)₂—, —OS(O)₂, —SP(O)(OR⁷)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, and—OP(O)(OR⁷)S—. In some embodiments, R¹ is selected from —OP(S)OR⁷)O—,—OP(O)(SR⁷)O—, and —OP(O)(OR⁷)S—. In some embodiments, R¹ is selectedfrom —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—, —OP(S)(O)O—, and —OP(O)(S⁻)O—. Insome embodiments. R¹ is selected from —OP(S)(OR⁷)O— and —OP(O)(SR⁷)O—.In some embodiments, R¹ is selected from —OP(O)(OR⁷)O—, —OP(OR⁷)N(R⁷)—,and —OPN(R⁷)₂O—. In some embodiments, R¹ is —OP(O)(OH)O—,—OP(O)(OCH₂CH₃)O—, —OP(OCH₂CH₂CN)N(CH(CH₃)₂)—, or —OPN(CH(CH₃)₂)₂O—. Insome embodiments, R¹ is selected from —OP(O)(OH)O— and OP(O)(O⁻)O—. Insome embodiments, R¹ comprises —O— or —S—. In some embodiments, R¹comprises —O—. In some embodiments, R¹ comprises —S—. In someembodiments, R¹ is a linker selected from —O— or —S—. In someembodiments, R¹ is —O—. In some embodiments, R¹ is —S—.

In some embodiments, each R² is independently selected from C₁₋₆alkyloptionally substituted with one or more substituents independentlyselected from halogen, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,—OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R² is independentlyselected from C₁₋₃ alkyl substituted with one or more substituentsindependently selected from halogen, —OR⁷, —OC(O)R⁷, —SR⁷, —N(R⁷)₂,—C(O)R⁷, and —S(O)R⁷. In some embodiments, each R² is independentlyselected from C₁₋₃ alkyl substituted with one or more substituentsindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R² is independently selected from C₁ alkyl substitutedwith one or more substituents independently selected from —OR⁷ and—OC(O)R⁷. In some embodiments, each R² is independently selected from—CH₂OH and —CH₂OC(O)CH₃.

In some embodiments, each R³ is independently selected from —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R³ is independently selected from halogen, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R³ isindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R³ is independently selected from —OR⁷ and —OC(O)R⁷.In some embodiments, R³ is independently selected from —OH and—OC(O)CH₃.

In some embodiments, each R⁴ is independently selected from —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R⁴ is independently selected from halogen, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R⁴ isindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R⁴ is independently selected from —OR⁷ and —OC(O)R⁷.In some embodiments, R⁴ is independently selected from —OH and—OC(O)CH₃.

In some embodiments, each R⁵ is independently selected from —OC(O)R⁷,—OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)R⁷,—C(O)OR⁷, and —C(O)N(R⁷)₂. In some embodiments, each R⁵ is selected from—OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, and—N(R⁷)C(O)OR⁷. In some embodiments, each R⁵ is independently selectedfrom —OC(O)R⁷ and —N(R⁷)C(O)R⁷. In some embodiments, each R⁵ isindependently selected from —N(H)C(O)CH₃.

In some embodiments, each R⁶ is independently selected from hydrogen,halogen, —CN, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and—S(O)R⁷; and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R⁶ is independently selected from hydrogen, halogen,—CN —OR⁷, —SR⁷, —N(R⁷)₂, and C₁₋₆ alkyl optionally substituted with oneor more substituents independently selected from halogen, —OR⁷, —SR⁷,and —N(R⁷)₁. In some embodiments, each R⁶ is independently selected fromhydrogen, halogen, —CN, —OH, —SH, and —NH₂.

In some embodiments, each R⁷ is independently selected from: hydrogen;C₁₋₄ alkyl, C₂₋₆ alkenyl, and C₂₋₄ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to10-membered heterocycle; and C₃₋₁₀ carbocycle, and 3- to 10-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,—NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆alkyl), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocycle, 3- to10-membered heterocycle, and C₁₋₆ haloalkyl. In some embodiments, eachR⁷ is independently selected from: hydrogen; and C₁₋₆ alkyl optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, 3- to10-membered heterocycle. In some embodiments, each R⁷ is independentlyselected from C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,—NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, and—NH(C₁₋₆ alkyl). In some embodiments, R⁷ is independently selected fromC₁₋₄ alkyl optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, and —SH. In someembodiments, each R⁷ is independently selected from hydrogen. In someembodiments, each R⁷ is independently selected from C₁₋₃ alkyloptionally substituted with one or more substituents independentlyselected from halogen, —CN, —OH, —SH, —NO₂, —NH₂, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, and —NH(C₁₋₆ alkyl).

In some embodiments, w is 1; v is 1; n is 2; m is 1 or 2; z is 3 and Yis C; Q is phenyl or cyclohexyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen, —CN,—OH, —SH, —NO₂, —NH₂, and C₁₋₃alkyl; R⁷ is selected from —OP(O)(OR⁷)O—,—OP(S)(OR⁷)O—, —OP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(OR⁷)O—;R² is C₁₋₃ alkyl substituted with —OH or —OC(O)CH₃; R³ is —OH or—OC(O)CH₃; R⁴ is —OH or —OC(O)CH₃; and R⁵ is —NH(O)CH₃.

In some embodiments, w is 1; v is 1; n is 2; m is 1 or 2; z is 3 and Yis C; Q is phenyl or cyclohexyl, each of which is optionally substitutedwith one or more substituents independently selected from halogen, —CN,—OH, —SH, —NO₂, —NH₂, and C₁₋₃ alkyl; R¹ is selected from —OP(O)(OH)O—,—OP(S)(OH)O—, —OP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(OH)O—;R² is C₁ alkyl substituted with —OH or —OC(O)CH₃; R³ is —OH or—OC(O)CH₃; R⁴ is —OH or —OC(O)CH₃; and R⁵ is —NH(O)CH₃. In someembodiments, w is 1; v is 1; n is 2; m is 1 or 2; z is 3 and Y is C; Qis phenyl or cyclohexyl, each of which is optionally substituted withone or more substituents independently selected from halogen, —CN, —OH,—SH, —NO₂, —NH₂, and C₁₋₃ alkyl; R¹ is selected from —OP(O)(OH)O—,—OP(S)(OH)O—, and —OP(OH)O—; R² is C₁ alkyl substituted with —OH or—OC(O)CH₃; R³ is —OH or —OC(O)CH₃; R⁴ is —OH or —OC(O)CH₃; and R⁵ is—NH(O)CH₃.

In some embodiments, a compound of Formula (I) is selected from:

In some embodiments, a compound of Formula (II) is selected from:

In some embodiments, the compound of Formula (I), (II), or (III) bindsto a lectin. In some embodiments, the compound binds to anasialoglycoprotein receptor. In some embodiments, the compound binds toa liver cell receptor. In some embodiments, the compound binds to ahepatocyte receptor. In some embodiments, the compound targets a livercell.

Provided herein, in some embodiments, compositions described hereincomprise a GalNAc compound. In some embodiments, a GalNAc compounddescribes a compound of Formula (A) or Formula (B):

-   -   or a salt thereof, wherein    -   each w is independently selected from any value from 1 to 20;    -   each v is independently selected from any value from 1 to 20;    -   n is selected from any value from 1 to 20;    -   m is selected from any value from 1 to 20;    -   z is selected from any value from 1 to 3, wherein        -   if z is 3, Y is C        -   if z is 2, Y is CR⁶, or        -   if z is 1, Y is C(R⁶)₂;    -   Q is selected from:        -   C₃₋₁₀ carbocycle optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl,            is optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            and —NH₂;    -   R¹ is selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, —S(O)R⁷, —S(O)₂R⁷, —OS(O)₂R⁷, —OP(O)(OR⁷)₂,            —OP(S)(OR⁷)₂, —SP(O)(OR⁷)₂, —OP(O)(SR⁷)(OR⁷),            —OP(O)(OR⁷)N(R⁷)₂, —OP(S)(OR⁷)N(R⁷)₂, —SP(O)(OR⁷)N(R⁷)₂,            —OP(O)(SR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(S)(N(R⁷)₂,            —SP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂, —SP(OR⁷)₂, —OP(OR⁷)(SR⁷),            —OP(OR⁷)N(R⁷)₂, —OP(SR⁷)N(R⁷)₂, —SP(OR⁷)N(R⁷)₂,            —OP(N(R⁷)₂)₂, and —SP(N(R⁷)₂)₂;    -   each R² is independently selected from:        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —OR⁷,            —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   R³ and R⁴ are each independently selected from:        -   —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁵ is independently selected from:        -   —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R)C(O)N(R⁷)₂,            —N(R⁷)C(O)OR⁷, —C(O)R⁷, —C(O)OR⁷, and —C(O)N(R⁷)₂;    -   each R⁶ is independently selected from:        -   hydrogen;        -   halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,            —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,            —N(R)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; and        -   C₁₋₆ alkyl optionally substituted with one or more            substituents independently selected from halogen, —CN, —NO₂,            —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,            —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,            —OC(O)R⁷, and —S(O)R⁷;    -   each R⁷ is independently selected from:        -   hydrogen;        -   C₁₋₆-alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is            optionally substituted with one or more substituents            independently selected from halogen, —CN, —OH, —SH, —NO₂,            —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂,            —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to 10-membered            heterocycle; and        -   C₃₋₁₀ carbocycle, and 3- to 10-membered heterocycle, each of            which is optionally substituted with one or more            substituents independently selected from halogen, —CN, —OH,            —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl,            —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₁₋₆ alkyl, C₂₋₆ alkenyl,            C₂₋₆ alkynyl, C₃₋₁₀ carbocycle, 3- to 10-membered            heterocycle, and C₁₋₆ haloalkyl.

In some embodiments, each w is independently selected from any valuefrom 1 to 20. In some embodiments, each w is independently selected fromany value from 1 to 15. In some embodiments, each w is independentlyselected from any value from 1 to 10. In some embodiments, each w isindependently selected from any value from 1 to 5. In some embodiments,each w is independently selected from any value from 1 to 4. In someembodiments, each w is independently selected from any value from 1 to3. In some embodiments, each w is independently selected from any valuefrom 1 to 2. In some embodiments, W is 1. In some embodiments, w is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments, each v is independently selected from any valuefrom 1 to 20. In some embodiments, each v is independently selected fromany value from 1 to 15. In some embodiments, each v is independentlyselected from any value from 1 to 10. In some embodiments, each v isindependently selected from any value from 1 to 5. In some embodiments,each v is independently selected from any value from 1 to 4. In someembodiments, each v is independently selected from any value from 1 to3. In some embodiments, each v is independently selected from any valuefrom 1 to 2. In some embodiments, each v is independently 1. In someembodiments, v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

In some embodiments, n is selected from any value from 1 to 20. In someembodiments, n is selected from any value from 1 to 15. In someembodiments, n is selected from any value from 1 to 10. In someembodiments, n is selected from any value from 1 to 9. In someembodiments, n is selected from any value from 1 to 8. In someembodiments, n is selected from any value from 1 to 7. In someembodiments, n is selected from any value from 1 to 6. In someembodiments, n is selected from any value from 1 to 5. In someembodiments, n is selected from any value from 1 to 4. In someembodiments, n is selected from any value from 2 to 4. In someembodiments, n is selected from any value from 1 to 3. In someembodiments, n is 2 or 3. In some embodiments, n is 3. In someembodiments, n is selected from any value from 1 to 2. In someembodiments, n is 2. In some embodiments, n is 1. In some embodiments, nis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20.

In some embodiments, m is selected from any value from 1 to 20. In someembodiments, m is selected from any value from 1 to 15. In someembodiments, m is selected from any value from 1 to 10. In someembodiments, m is selected from any value from 1 to 9. In someembodiments, m is selected from any value from 1 to 8. In someembodiments, m is selected from any value from 1 to 7. In someembodiments, m is selected from any value from 3 to 7. In someembodiments, m is selected from any value from 1 to 6. In someembodiments, m is selected from any value from 2 to 6. In someembodiments, m is selected from any value from 3 to 6. In someembodiments, m is selected from any value from 4 to 6. In someembodiments, m is 6. In some embodiments, m is selected from any valuefrom 1 to 5. In some embodiments, m is selected from any value from 3 to5. In some embodiments, m is 5. In some embodiments, m is 4 or 5. Insome embodiments, m is selected from any value from 1 to 4. In someembodiments, m is 4. In some embodiments, m is 3 or 4. In someembodiments, m is selected from any value from 2 to 4. In someembodiments, m is selected from any value from 1 to 3. In someembodiments, m is 3. In some embodiments, m is selected from any valuefrom 1 to 2. In some embodiments, m is 2. In some embodiments, m is 1.In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

In some embodiments, z is selected from any value from 1 to 3. In someembodiments, z is 3 and Y is C. In some embodiments, z is 2 and Y isCR⁶. In some embodiments, z is 1 and Y is C(R⁶)₂.

In some embodiments, Q is selected from C₃₋₁₀ carbocycle optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R)₂,—N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,—OC(O)R⁷, —S(O)R⁷, and C₁₋₆ alkyl, wherein the C₁₋₆ alkyl, is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, and —NH₂. In some embodiments. Q isselected from C₁₋₆ carbocycle optionally substituted with one or moresubstituents independently selected from halogen, —CN, —NO₂, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, —S(O)R⁷, and C₁₋₆alkyl, wherein the C₁₋₆ alkyl, is optionally substituted with one ormore substituents independently selected from halogen, —CN, —OH, —SH,—NO₂, and —NH₂. In some embodiments, Q is selected from C₁₋₆ carbocycleoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, Qis selected from C₆ carbocycle optionally substituted with one or moresubstituents independently selected from halogen, —CN, —NO₂, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, Q is selected from C₅ carbocycle optionally substitutedwith one or more substituents independently selected from halogen, —CN,—NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and—S(O)R⁷. In some embodiments, Q is selected from C₅₋₆ carbocycleoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. In someembodiments, Q is selected from phenyl, cyclohexyl, cyclopentadiene, andcyclopentyl, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,and —NH₂. In some embodiments, Q is selected from phenyl and cyclohexyl,each of which is optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂. Insome embodiments, Q is selected from phenyl and cyclohexyl. In someembodiments, Q is phenyl. In some embodiments, Q is cyclohexyl.

In some embodiments, R¹ is selected from —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, —S(O)R⁷, —S(O)₂R⁷, —OS(O)₂R⁷,—OP(O)(OR⁷)₂, —OP(S)(OR⁷)₂, —SP(O)(OR⁷)₂, —OP(O)(SR⁷)(OR⁷),—OP(O)(OR⁷)N(R⁷)₂, —OP(S)(OR⁷)N(R⁷)₂, —SP(O)(OR⁷)N(R⁷)₂,—OP(O)(SR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(S)(N(R⁷)₂)₂, —SP(O)(N(R⁷)₂)₂,—OP(OR⁷)₂, —SP(OR⁷)₂, —OP(OR⁷)(SR⁷), —OP(OR⁷)N(R⁷)₂, —OP(SR⁷)N(R⁷)₂,—SP(OR⁷)N(R⁷)₂, —OP(N(R⁷)₂)₂, and —SP(N(R⁷)₂)₂. In some embodiments. R¹is selected from —OR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷,—OP(O)(OR⁷)₂, —OP(O)(OR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂,—OP(OR⁷)N(R⁷)₂, and —OP(N(R⁷)₂)₂. In some embodiments, R¹ is selectedfrom —SR⁷, —S(O)R⁷, —S(O)₂R⁷, —OS(O)₂R⁷, —OP(S)(OR⁷)₂, —SP(O)(OR⁷)₂,—OP(O)(SR⁷)(OR⁷), —OP(S)(OR⁷)N(R⁷)₂, —SP(O)(OR⁷)N(R⁷)₂,—OP(O)(SR⁷)N(R⁷)₂, —OP(S)(N(R⁷)₂)₂, —SP(O)(N(R⁷)₂)₂, —SP(OR⁷)₂,—OP(OR⁷)(SR⁷), —OP(SR⁷)N(R⁷)₂, —SP(OR⁷)N(R⁷)₂, and —SP(N(R⁷)₂)₂. In someembodiments, R¹ is selected from —OP(O)(OR⁷)₂, —OP(S)(OR⁷)₂,—SP(O)(OR⁷)₂, —OP(O)(SR⁷)(OR⁷), —OP(O)(OR⁷)N(R⁷)₂, —OP(S)(OR⁷)N(R⁷)₂,—SP(O)(OR⁷)N(R⁷)₂, —OP(O)(SR⁷)N(R⁷)₂, —OP(O)(N(R⁷)₂)₂, —OP(S)(N(R⁷)₂)₂,—SP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂, —SP(OR⁷)₂, —OP(OR⁷)(SR⁷), —OP(OR⁷)N(R⁷)₂,—OP(SR⁷)N(R⁷)₂, —SP(OR⁷)N(R⁷)₂, —OP(N(R⁷)₂)₂, and —SP(N(R⁷)₂)₂. In someembodiments, R¹ is a selected from —OP(O)(OR⁷)₂, —OP(O)(OR⁷)N(R⁷)₂,—OP(O)(N(R⁷)₂)₂, —OP(OR⁷)₂, —OP(OR⁷)N(R⁷)₂, and —OP((NR⁷)₂)₂. In someembodiments, R¹ is a selected from —OP(O)(OR⁷)₂ and —OP(OR⁷)N(R⁷)₂. Insome embodiments, R¹ is selected from —OP(O)(OCH₂CH₃)OH and—OP(OCH₂CH₂CN)N(CH(CH₃)₂)₂. In some embodiments, R¹ is—OP(OCH₂CH₂CN)N(CH(CH₃)₂)₂.

In some embodiments, each R² is independently selected from C₁₋₆alkyloptionally substituted with one or more substituents independentlyselected from halogen, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂,—N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷,—OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R² is independentlyselected from C₁₋₃ alkyl substituted with one or more substituentsindependently selected from halogen, —OR⁷, —OC(O)R⁷, —SR⁷, —N(R⁷)₂,—C(O)R⁷, and —S(O)R⁷. In some embodiments, each R² is independentlyselected from C₁₋₃ alkyl substituted with one or more substituentsindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R² is independently selected from C₁ alkyl substitutedwith one or more substituents independently selected from —OR⁷ and—OC(O)R⁷. In some embodiments, each R² is independently selected from—CH₂OH and —CH₂OC(O)CH₃.

In some embodiments, each R³ is independently selected from —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R¹ is independently selected from halogen, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R³ isindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R³ is independently selected from —OR⁷ and —OC(O)R⁷.In some embodiments, R³ is independently selected from —OH and—OC(O)CH₃.

In some embodiments, each R⁴ is independently selected from —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R⁴ is independently selected from halogen, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —OC(O)R⁷, and —S(O)R⁷. In some embodiments, each R⁴ isindependently selected from —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂. In someembodiments, each R⁴ is independently selected from —OR⁷ and —OC(O)R⁷.In some embodiments. R⁴ is independently selected from —OH and—OC(O)CH₃.

In some embodiments, each R⁵ is independently selected from —OC(O)R⁷,—OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R)₂, —N(R⁷)C(O)OR⁷, —C(O)R⁷,—C(O)OR⁷, and —C(O)N(R⁷)₂. In some embodiments, each R⁵ is selected from—OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, and—N(R⁷)C(O)OR⁷. In some embodiments, each R⁵ is independently selectedfrom —OC(O)R⁷ and —N(R⁷)C(O)R⁷. In some embodiments, each R⁵ isindependently selected from —N(H)C(O)CH₃.

In some embodiments, each R⁶ is independently selected from hydrogen,halogen, —CN, —OR⁷, —SR, —N(R⁷)₂, —C(O)R, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and—S(O)R⁷; and C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷. In someembodiments, each R⁶ is independently selected from hydrogen, halogen,—CN —OR⁷, —SR⁷, —N(R⁷)₂, and C₁₋₆ alkyl optionally substituted with oneor more substituents independently selected from halogen, —OR⁷, —SR⁷,and —N(R⁷)₂. In some embodiments, each R⁶ is independently selected fromhydrogen, halogen, —CN, —OH, —SH, and —NH₂.

In some embodiments, each R⁷ is independently selected from: hydrogen;C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, and 3- to10-membered heterocycle; and C₃₋₁₀ carbocycle, and 3- to 10-memberedheterocycle, each of which is optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,—NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₄ alkyl)₂, —NH(C₁₋₆alkyl), C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₄ alkynyl, C₃₋₁₀ carbocycle, 3- to10-membered heterocycle, and C₁₋₆ haloalkyl. In some embodiments, eachR⁷ is independently selected from: hydrogen; and C₁₋₆ alkyl optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀ carbocycle, 3- to10-membered heterocycle. In some embodiments, each R⁷ is independentlyselected from C₁₋₆ alkyl optionally substituted with one or moresubstituents independently selected from halogen, —CN, —OH, —SH, —NO₂,—NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, and—NH(C₁₋₆ alkyl). In some embodiments, each R⁷ is independently selectedfrom hydrogen. In some embodiments, each R⁷ is independently selectedfrom C₁₋₃ alkyl optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, —SH, —NO₂, —NH₂, —O—C₁₋₆alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, and —NH(C₁₋₆ alkyl). In someembodiments, each R⁷ is independently selected from C₁₋₆ alkyloptionally substituted with one or more substituents independentlyselected from halogen, —CN, —OH, and —SH.

In some embodiments, a compound of Formula (A) is selected from:

Provided herein, in some embodiments, is a compound represented byFormula (B):

In some embodiments, the phosphate is deprotonated to form a salt ofFormula (I), (II), (A), or (B). In some embodiments, the cation is ametal ion such as a metal cation. Non limiting examples of metal cationsinclude Na⁺, K⁺, Mg²⁺, and Ca²⁺. In some embodiments, the metal cationcomprises Na. In some embodiments, the metal cation comprises K. In someembodiments, the metal cation comprises Mg²⁺. In some embodiments, themetal cation comprises Ca²⁺. In some embodiments, the cation is anorganic or inorganic small molecule. In some embodiments wherein thecompound is a deprotonated form of Formula (I) or (II), the cation is apositively charged nucleic acid present in the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more additional phosphates, or one or morephosphorothioates linking to the oligonucleotide. J may include one ormore additional phosphates linking to the oligonucleotide. J may includeone or more phosphorothioates linking to the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more additional phosphates, or one or morephosphorothioates linking to the oligonucleotide. J may include one ormore additional phosphates linking to the oligonucleotide. J may includeone or more phosphorothioates linking to the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more phosphates or phosphorothioates linking to theoligonucleotide. J may include one or more phosphates linking to theoligonucleotide. J may include a phosphate linking to theoligonucleotide. J may include one or more phosphorothioates linking tothe oligonucleotide. J may include a phosphorothioate linking to theoligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

The structure in this compound attached to the oligonucleotide (J) is anexample of a GalNAc moiety. J may include one or more phosphates orphosphorothioates linking to the oligonucleotide. J may include one ormore phosphates linking to the oligonucleotide. J may include aphosphate linking to the oligonucleotide. J may include one or morephosphorothioates linking to the oligonucleotide. J may include aphosphorothioate linking to the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more additional phosphates, or one or morephosphorothioates linking to the oligonucleotide. J may include one ormore additional phosphates linking to the oligonucleotide. J may includeone or more phosphorothioates linking to the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more additional phosphates, or one or morephosphorothioates linking to the oligonucleotide. J may include one ormore additional phosphates linking to the oligonucleotide. J may includeone or more phosphorothioates linking to the oligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

J may include one or more phosphates or phosphorothioates linking to theoligonucleotide. J may include one or more phosphates linking to theoligonucleotide. J may include a phosphate linking to theoligonucleotide. J may include one or more phosphorothioates linking tothe oligonucleotide. J may include a phosphorothioate linking to theoligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

The structure in this compound attached to the oligonucleotide (J) maybe referred to as “ETL17,” and is an example of a GalNAc moiety. J mayinclude one or more phosphates or phosphorothioates linking to theoligonucleotide. J may include one or more phosphates linking to theoligonucleotide. J may include a phosphate linking to theoligonucleotide. J may include one or more phosphorothioates linking tothe oligonucleotide. J may include a phosphorothioate linking to theoligonucleotide.

Some embodiments include the following, where J is the oligonucleotide:

The structure in this compound attached to the oligonucleotide (J) is anexample of a GalNAc moiety. J may include one or more phosphates orphosphorothioates linking to the oligonucleotide. J may include one ormore phosphates linking to the oligonucleotide. J may include aphosphate linking to the oligonucleotide. J may include one or morephosphorothioates linking to the oligonucleotide. J may include aphosphorothioate linking to the oligonucleotide. In any embodiment, Jmay include an additional linker.

1. Analogues

Chemical entities having carbon-carbon double bonds or carbon-nitrogendouble bonds may exist in Z- or E-form (or cis- or trans-form).Furthermore, some chemical entities may exist in various tautomericforms. Unless otherwise specified, compounds described herein areintended to include all Z-, E- and tautomeric forms as well.

A “tautomer” refers to a molecule or moiety wherein a proton shift fromone atom of a molecule to another atom of the same molecule is possible.The compounds presented herein, in certain embodiments, exist astautomers. In circumstances where tautomerization is possible, achemical equilibrium of the tautomers will exist. The exact ratio of thetautomers depends on several factors, including physical state,temperature, solvent, and pH. Some examples of tautomeric equilibriuminclude:

The compounds and moieties disclosed herein, in some embodiments, areused in different enriched isotopic forms, e.g., enriched in the contentof ²H, ³H, ¹¹C, ¹³C and/or ¹⁴C. In one particular embodiment, thecompound is deuterated in at least one position. Such deuterated formscan be made by the procedure described in U.S. Pat. Nos. 5,846,514 and6,334,997. As described in U.S. Pat. Nos. 5,846,514 and 6,334,997,deuteration can improve the metabolic stability and or efficacy, thusincreasing the duration of action of drugs.

Unless otherwise stated, compounds described herein and moieties areintended to include compounds and moieties which differ only in thepresence of one or more isotopically enriched atoms. For example,compounds having the present structures except for the replacement of ahydrogen by a deuterium or tritium, or the replacement of a carbon by¹³C- or ¹⁴C-enriched carbon are within the scope of the presentdisclosure.

The compounds and moieties of the present disclosure optionally containunnatural proportions of atomic isotopes at one or more atoms thatconstitute such compounds. For example, the compounds may be labeledwith isotopes, such as for example, deuterium (²H), tritium (³H),iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). Isotopic substitution with ²H,¹¹C, ¹³C, ¹⁴C, ¹⁵C, ¹²N, ¹³N, ¹⁵N, ¹⁶N, ¹⁶O, ¹⁷O, ¹⁴F, ¹⁵F, ¹⁶F, ¹⁷F,¹⁸F, ³³S, ³⁴S, ³⁵S, ³⁶S, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br, and ¹²⁵I are allcontemplated. All isotopic variations of the compounds and moieties ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

In certain embodiments, the compounds and moieties disclosed herein havesome or all of the ¹H atoms replaced with ²H atoms. The methods ofsynthesis for deuterium-containing compounds are known in the art andinclude, by way of non-limiting example only, the following syntheticmethods.

Deuterium substituted compounds are synthesized using various methodssuch as described in: Dean, Dennis C.; Editor. Recent Advances in theSynthesis and Applications of Radiolabeled Compounds for Drug Discoveryand Development. [In: Cuff., Pharm. Des., 2000; 6(10)] 2000. ¹¹⁰ pp;George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compoundsvia Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21;and Evans. E. Anthony. Synthesis of radiolabeled compounds. J.Radioanal. Chem., 1981, 64(1-2), 9-32.

Deuterated starting materials are readily available and are subjected tothe synthetic methods described herein to provide for the synthesis ofdeuterium-containing compounds. Large numbers of deuterium-containingreagents and building blocks are available commercially from chemicalvendors, such as Aldrich Chemical Co.

Included in the present disclosure are salts, particularlypharmaceutically acceptable salts, of the compounds described herein.The compounds of the present disclosure that possess a sufficientlyacidic, a sufficiently basic, or both functional groups, can react withany of a number of inorganic bases, and inorganic and organic acids, toform a salt. Alternatively, compounds that are inherently charged, suchas those with a quaternary nitrogen, can form a salt with an appropriatecounterion, e.g., a halide such as bromide, chloride, or fluoride,particularly bromide.

The compounds described herein may in some cases exist as diastereomers,enantiomers, or other stereoisomeric forms. The compounds presentedherein include all diastereomeric, enantiomeric, and epimeric forms aswell as the appropriate mixtures thereof. Separation of stereoisomersmay be performed by chromatography or by forming diastereomers andseparating by recrystallization, or chromatography, or any combinationthereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers,Racemates and Resolutions”, John Wiley And Sons, Inc., 1981, hereinincorporated by reference for this disclosure). Stereoisomers may alsobe obtained by stereoselective synthesis.

The methods and compositions described herein include the use ofamorphous forms as well as crystalline forms (also known as polymorphs).The compounds described herein may be in the form of pharmaceuticallyacceptable salts. As well, in some embodiments, active metabolites ofthese compounds having the same type of activity are included in thescope of the present disclosure. In addition, the compounds describedherein can exist in unsolvated or solvated forms with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. The solvatedforms of the compounds presented herein are also considered to bedisclosed herein.

B. Oligonucleotides

Provided herein, in some embodiments, are compositions or compoundscomprising an oligonucleotide. The oligonucleotide may be conjugated toa GalNAc moiety. The oligonucleotide may be directly connected to alinker connected to the GalNAc moiety. The oligonucleotide may be usedin a method described herein.

In some embodiments, the oligonucleotide binds to a targetoligonucleotide. Examples of target oligonucleotides include a targetRNA or a target DNA. In some embodiments, the oligonucleotide binds to atarget DNA. In some embodiments, the oligonucleotide binds to a targetDNA, and inhibits RNA (e.g. mRNA) expression from the target DNA. Insome embodiments, the oligonucleotide binds to a target RNA. The targetRNA may include a target mRNA. In some embodiments, the oligonucleotidebinds to a target mRNA. In some embodiments, the oligonucleotideinhibits the target mRNA such as by reducing an amount of the targetmRNA, causing degradation of the target mRNA, or decreasing orpreventing translation of the target mRNA. In some embodiments, theoligonucleotide reduces an amount of a target protein produced from thetarget mRNA, for example by inhibiting the target mRNA. Theoligonucleotide may include a small interfering RNA (siRNA). Theoligonucleotide may include an antisense oligonucleotide (ASO).

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide and inhibits expression of a targetprotein encoded by the target oligonucleotide. In some embodiments, thecomposition comprises an oligonucleotide that binds to a target RNA andinhibits expression of a target protein encoded by the target RNA. Insome embodiments, the composition comprises an oligonucleotide thatbinds to a target mRNA and inhibits expression of a target proteinencoded by the target mRNA.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide and inhibits expression of a secondoligonucleotide encoded by the target oligonucleotide. In someembodiments, the composition comprises an oligonucleotide that binds toa target DNA and inhibits expression of a target RNA encoded by thetarget DNA. In some embodiments, the composition comprises anoligonucleotide that binds to a target DNA and inhibits expression of atarget mRNA encoded by the target DNA.

Target oligonucleotides may be identified by a variety of ways. In someinstances, the target oligonucleotide comprises an mRNA that hasexpression levels that are associated with incidence of a disorder (e.g.a liver disorder). In some instances, the target oligonucleotidecomprises an mRNA that is encoded by a gene that has a particulargenotype associated with the disorder. Large-scale human genetic datacan improve the success rate of pharmaceutical discovery anddevelopment. A Genome Wide Association Study (GWAS) may detectassociations between genetic variants and traits in a population sample.A GWAS may enable better understanding of the biology of disease, andprovide applicable treatments. A GWAS can utilize genotyping and/orsequencing data, and often involves an evaluation of millions of geneticvariants that are relatively evenly distributed across the genome. Themost common GWAS design is the case-control study, which involvescomparing variant frequencies in cases versus controls. If a variant hasa significantly different frequency in cases versus controls, thatvariant is said to be associated with disease. Association statisticsthat may be used in a GWAS are p-values, as a measure of statisticalsignificance; odds ratios (OR), as a measure of effect size; or betacoefficients (beta), as a measure of effect size. Researchers oftenassume an additive genetic model and calculate an allelic odds ratio,which is the increased (or decreased) risk of disease conferred by eachadditional copy of an allele (compared to carrying no copies of thatallele). An additional concept in design and interpretation of GWAS isthat of linkage disequilibrium, which is the non-random association ofalleles. The presence of linkage disequilibrium can obfuscate whichvariant is “causal.”

Functional annotation of variants and/or wet lab experimentation canidentify the causal genetic variant identified via GWAS, and in manycases may lead to the identification of disease-causing genes. Inparticular, understanding the functional effect of a causal geneticvariant (for example, loss of protein function, gain of proteinfunction, increase in gene expression, or decrease in gene expression)may allow that variant to be used as a proxy for therapeutic modulationof the target gene, or to gain insight into potential therapeuticefficacy and safety of a therapeutic that modulates that target.

Identification of such gene-disease associations has provided insightsinto disease biology and may be used to identify novel therapeutictargets for the pharmaceutical industry. In order to translate thetherapeutic insights derived from human genetics, disease biology inpatients may be exogenously ‘programmed’ into replicating theobservation from human genetics. There are several potential options fortherapeutic modalities that may be brought to bear in translatingtherapeutic targets identified via human genetics into novel medicines.These may include well established therapeutic modalities such as smallmolecules and monoclonal antibodies, maturing modalities such asoligonucleotides, and emerging modalities such as gene therapy and geneediting. The choice of therapeutic modality can depend on severalfactors including the location of a target (for example, intracellular,extracellular, or secreted), a relevant tissue (for example, liver) anda relevant indication. Such studies may be conducted to identifyspecific liver disorder-related targets for siRNA or ASO inhibition by acomposition or compound described herein.

Some embodiments include a method of making an oligonucleotide or siRNAusing a method disclosed herein. For example, any aspect of an Exampleherein that includes steps for synthesis may be used. Some embodimentsinclude making a GalNAc moiety, or making an oligonucleotide with aGalNAc moiety.

1. siRNAs

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide (e.g. mRNA), wherein theoligonucleotide comprises a small interfering RNA (siRNA). In someembodiments, the composition comprises an oligonucleotide that binds toa target oligonucleotide (e.g. mRNA), wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand. Insome embodiments, the sense strand comprises RNA. In some embodiments,the antisense strand comprises RNA.

In some embodiments, the sense strand is 12-30 nucleosides in length. Insome embodiments, the sense strand is 14-30 nucleosides in length. Insome embodiments, the sense strand is at least about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleosides in length. In some embodiments, the sense strand is at least12 nucleotides in length. In some embodiments, the sense strand is atleast 14 nucleotides in length. In some embodiments, the sense strand isat least 16 nucleotides in length. In some embodiments, the sense strandis at least 18 nucleotides in length. In some embodiments, the sensestrand is at least 20 nucleotides in length. In some embodiments, thesense strand is at least 22 nucleotides in length. In some embodiments,the sense strand is at least 24 nucleotides in length. In someembodiments, the sense strand is at least 26 nucleotides in length. Insome embodiments, the sense strand is at least 28 nucleotides in length.In some embodiments, the sense strand is at least 30 nucleotides inlength. In some embodiments, the sense strand is no more than about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 nucleosides in length. In some embodiments, the sense strandis no more than 12 nucleotides in length. In some embodiments, the sensestrand is no more than 14 nucleotides in length. In some embodiments,the sense strand is no more than 16 nucleotides in length. In someembodiments, the sense strand is no more than 18 nucleotides in length.In some embodiments, the sense strand is no more than 20 nucleotides inlength. In some embodiments, the sense strand is no more than 22nucleotides in length. In some embodiments, the sense strand is no morethan 24 nucleotides in length. In some embodiments, the sense strand isno more than 26 nucleotides in length. In some embodiments, the sensestrand is no more than 28 nucleotides in length. In some embodiments,the sense strand is no more than 30 nucleotides in length.

In some embodiments, the antisense strand is 12-30 nucleosides inlength. In some embodiments, the antisense strand is 14-30 nucleosidesin length. In some embodiments, the antisense strand is at least about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleosides in length. In some embodiments, the antisensestrand is at least 12 nucleotides in length. In some embodiments, theantisense strand is at least 14 nucleotides in length. In someembodiments, the antisense strand is at least 16 nucleotides in length.In some embodiments, the antisense strand is at least 18 nucleotides inlength. In some embodiments, the antisense strand is at least 20nucleotides in length. In some embodiments, the antisense strand is atleast 22 nucleotides in length. In some embodiments, the antisensestrand is at least 24 nucleotides in length. In some embodiments, theantisense strand is at least 26 nucleotides in length. In someembodiments, the antisense strand is at least 28 nucleotides in length.In some embodiments, the antisense strand is at least 30 nucleotides inlength. In some embodiments, the antisense strand is no more than about10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleosides in length. In some embodiments, the antisensestrand is no more than 12 nucleotides in length. In some embodiments,the antisense strand is no more than 14 nucleotides in length. In someembodiments, the antisense strand is no more than 16 nucleotides inlength. In some embodiments, the antisense strand is no more than 18nucleotides in length. In some embodiments, the antisense strand is nomore than 20 nucleotides in length. In some embodiments, the antisensestrand is no more than 22 nucleotides in length. In some embodiments,the antisense strand is no more than 24 nucleotides in length. In someembodiments, the antisense strand is no more than 26 nucleotides inlength. In some embodiments, the antisense strand is no more than 28nucleotides in length. In some embodiments, the antisense strand is nomore than 30 nucleotides in length. In some embodiments, the antisensestrand is the same length as the sense strand.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target oligonucleotide (e.g. mRNA), whereinthe oligonucleotide comprises an siRNA comprising a sense strand and anantisense strand, wherein the sense strand is 12-30 nucleosides inlength. In some embodiments, the composition comprises a sense strangethat is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleosides in length, or a range defined by anyof the two aforementioned numbers. In some embodiments, the compositioncomprises an antisense strand is 12-30 nucleosides in length. In someembodiments, the composition comprises an antisense strange that is 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 nucleosides in length, or a range defined by any of the twoaforementioned numbers.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target oligonucleotide (e.g. mRNA), whereinthe oligonucleotide comprises an siRNA comprising a sense strand and anantisense strand, each strand is independently about 14-30 nucleosidesin length, and at least one of the sense strand and the antisense strandcomprises a nucleoside sequence comprising about 12-30 contiguousnucleosides of a full-length human target mRNA sequence. In someembodiments, at least one of the sense strand and the antisense strandcomprise a nucleoside sequence comprising at least about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, ormore contiguous nucleosides of one of the full-length human target mRNAsequence.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand,wherein the sense strand and the antisense strand form a double-strandedRNA duplex. In some embodiments, the first base pair of thedouble-stranded RNA duplex is an AU base pair.

In some embodiments, the sense strand further comprises a 3′ overhang.In some embodiments, the 3′ overhang comprises 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleosides, or a range of nucleotides defined by any two ofthe aforementioned numbers. In some embodiments, the 3′ overhangcomprises 1, 2, or more nucleosides. In some embodiments, the 3′overhang comprises 2 nucleosides. In some embodiments, the sense strandfurther comprises a 5′ overhang. In some embodiments, the 5′ overhangcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or a range ofnucleotides defined by any two of the aforementioned numbers. In someembodiments, the 5′ overhang comprises 1, 2, or more nucleosides. Insome embodiments, the 5′ overhang comprises 2 nucleosides.

In some embodiments, the antisense strand further comprises a 3′overhang. In some embodiments, the 3′ overhang comprises 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleosides, or a range of nucleotides defined by anytwo of the aforementioned numbers. In some embodiments, the 3′ overhangcomprises 1, 2, or more nucleosides. In some embodiments, the 3′overhang comprises 2 nucleosides. In some embodiments, the antisensestrand further comprises a 5′ overhang. In some embodiments, the 5′overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, or arange of nucleotides defined by any two of the aforementioned numbers.In some embodiments, the 5′ overhang comprises 1, 2, or morenucleosides. In some embodiments, the 5′ overhang comprises 2nucleosides.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand,wherein the siRNA binds with a 19mer in a human target mRNA encoding thetarget protein. In some embodiments, the siRNA binds with a 12mer, a13mer, a 14mer, a 15mer, a 16mer, a 17mer, a 18mer, a 19mer, a 20mer, a21mer, a 22mer, a 23mer, a 24mer, or a 25mer in a human target mRNA.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand,wherein the siRNA binds with a 17mer in a non-human primate target mRNAencoding the target protein. In some embodiments, the siRNA binds with a12mer, a 13mer, a 14mer, a 15mer, a 16mer, a 17mer, a 18mer, a 19mer, a20mer, a 21mer, a 22mer, a 23mer, a 24mer, or a 25mer in a non-humanprimate target mRNA.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand,wherein the siRNA binds with a 19mer in a human target mRNA encoding thetarget protein, or a combination thereof. In some embodiments, the siRNAbinds with a 12mer, a 13mer, a 14mer, a 15mer, a 16mer, a 17mer, and18mer, a 19mer, a 20mer, a 21mer, a 22mer, a 23mer, a 24mer, or a 25merin a human target mRNA.

2. ASOs

In some embodiments, the composition comprises an oligonucleotide thatinhibits expression of a target oligonucleotide (e.g. mRNA), wherein theoligonucleotide comprises an antisense oligonucleotide (ASO). In someembodiments, the ASO is 12-30 nucleosides in length. In someembodiments, the ASO is 14-30 nucleosides in length. In someembodiments, the ASO is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length, or a rangedefined by any of the two aforementioned numbers. In some embodiments,the ASO is 15-25 nucleosides in length. In some embodiments, the ASO is20 nucleosides in length. In some embodiments, the ASO comprises DNA.

In some embodiments, the ASO is 12-30 nucleosides in length. In someembodiments, the ASO is 14-30 nucleosides in length. In someembodiments, the ASO is at least about 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides inlength. In some embodiments, the ASO is at least 12 nucleotides inlength. In some embodiments, the ASO is at least 14 nucleotides inlength. In some embodiments, the ASO is at least 16 nucleotides inlength. In some embodiments, the ASO is at least 18 nucleotides inlength. In some embodiments, the ASO is at least 20 nucleotides inlength. In some embodiments, the ASO is at least 22 nucleotides inlength. In some embodiments, the ASO is at least 24 nucleotides inlength. In some embodiments, the ASO is at least 26 nucleotides inlength. In some embodiments, the ASO is at least 28 nucleotides inlength. In some embodiments, the ASO is at least 30 nucleotides inlength. In some embodiments, the ASO is no more than about 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleosides in length. In some embodiments, the ASO is no more than12 nucleotides in length. In some embodiments, the ASO is no more than14 nucleotides in length. In some embodiments, the ASO is no more than16 nucleotides in length. In some embodiments, the ASO is no more than18 nucleotides in length. In some embodiments, the ASO is no more than20 nucleotides in length. In some embodiments, the ASO is no more than22 nucleotides in length. In some embodiments, the ASO is no more than24 nucleotides in length. In some embodiments, the ASO is no more than26 nucleotides in length. In some embodiments, the ASO is no more than28 nucleotides in length. In some embodiments, the ASO is no more than30 nucleotides in length.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an ASO about 12-30 nucleosides in length and comprising anucleoside sequence complementary to about 12-30 contiguous nucleosidesof a full-length human pre-mRNA target sequence encoding the targetprotein: wherein (i) the oligonucleotide comprises a modificationcomprising a modified nucleoside and/or a modified internucleosidelinkage, and/or (ii) the composition comprises a pharmaceuticallyacceptable carrier.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target protein, wherein the oligonucleotidecomprises an ASO about 12-30 nucleosides in length and comprising anucleoside sequence complementary to about 12-30 contiguous nucleosidesof a full-length human target mRNA sequence encoding the target protein;wherein (i) the oligonucleotide comprises a modification comprising amodified nucleoside and/or a modified internucleoside linkage, and/or(ii) the composition comprises a pharmaceutically acceptable carrier.

1. Oligonucleotide Modification Patterns

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesa modification comprising a modified nucleoside and/or a modifiedinternucleoside linkage, and/or (ii) the composition comprises apharmaceutically acceptable carrier. In some embodiments, theoligonucleotide comprises a modification comprising a modifiednucleoside and/or a modified internucleoside linkage. In someembodiments, the oligonucleotide comprises a modified internucleosidelinkage. In some embodiments, the modified internucleoside linkagecomprises alkylphosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, or carboxymethyl ester, or acombination thereof. In some embodiments, the modified internucleosidelinkage comprises one or more phosphorothioate linkages. Benefits of themodified internucleoside linkage may include decreased toxicity orimproved pharmacokinetics.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesa modified internucleoside linkage, wherein the oligonucleotidecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 modified internucleoside linkages, or a range of modifiedinternucleoside linkages defined by any two of the aforementionednumbers. In some embodiments, the oligonucleotide comprises no more than18 modified internucleoside linkages. In some embodiments, theoligonucleotide comprises no more than 20 modified internucleosidelinkages. In some embodiments, the oligonucleotide comprises 2 or moremodified internucleoside linkages, 3 or more modified internucleosidelinkages, 4 or more modified internucleoside linkages, 5 or moremodified internucleoside linkages, 6 or more modified internucleosidelinkages, 7 or more modified internucleoside linkages, 8 or moremodified internucleoside linkages, 9 or more modified internucleosidelinkages, 10 or more modified internucleoside linkages, 11 or moremodified internucleoside linkages, 12 or more modified internucleosidelinkages, 13 or more modified internucleoside linkages, 14 or moremodified internucleoside linkages, 15 or more modified internucleosidelinkages, 16 or more modified internucleoside linkages, 17 or moremodified internucleoside linkages, 18 or more modified internucleosidelinkages, 19 or more modified internucleoside linkages, or 20 or moremodified internucleoside linkages.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesthe modified nucleoside. In some embodiments, the modified nucleosidecomprises a locked nucleic acid (LNA), hexitol nucleic acid (HLA),cyclohexene nucleic acid (CeNA), 2′-methoxyethyl, 2′-O-alkyl,2′-O-allyl, 2′-fluoro, or 2′-deoxy, or a combination thereof. In someembodiments, the modified nucleoside comprises a LNA. In someembodiments, the modified nucleoside comprises a 2′,4′ constrained ethylnucleic acid. In some embodiments, the modified nucleoside comprisesHLA. In some embodiments, the modified nucleoside comprises CeNA. Insome embodiments, the modified nucleoside comprises a 2′-methoxyethylgroup. In some embodiments, the modified nucleoside comprises a2′-O-alkyl group. In some embodiments, the modified nucleoside comprisesa 2′-O-allyl group. In some embodiments, the modified nucleosidecomprises a 2′-fluoro group. In some embodiments, the modifiednucleoside comprises a 2′-deoxy group. In some embodiments, the modifiednucleoside comprises a 2′-O-methyl nucleoside, 2′-deoxyfluoronucleoside, 2′-O—N-methylacetamido (2′-O-NMA) nucleoside, a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleoside, 2′-O-aminopropyl(2′-O-AP) nucleoside, or 2′-ara-F, or a combination thereof. In someembodiments, the modified nucleoside comprises a 2′-O-methyl nucleoside.In some embodiments, the modified nucleoside comprises a 2′-deoxyfluoronucleoside. In some embodiments, the modified nucleoside comprises a2′-O-NMA nucleoside. In some embodiments, the modified nucleosidecomprises a 2′-O-DMAEOE nucleoside. In some embodiments, the modifiednucleoside comprises a 2′-O-aminopropyl (2′-O-AP) nucleoside. In someembodiments, the modified nucleoside comprises 2′-ara-F. In someembodiments, the modified nucleoside comprises one or more 2′fluoromodified nucleosides. In some embodiments, the modified nucleosidecomprises a 2′ O-alkyl modified nucleoside. Benefits of the modifiednucleoside may include decreased toxicity or improved pharmacokinetics.

In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 modifiednucleosides, or a range of nucleosides defined by any two of theaforementioned numbers. In some embodiments, the oligonucleotidecomprises no more than 19 modified nucleosides. In some embodiments, theoligonucleotide comprises no more than 21 modified nucleosides. In someembodiments, the oligonucleotide comprises 2 or more modifiednucleosides, 3 or more modified nucleosides, 4 or more modifiednucleosides, 5 or more modified nucleosides, 6 or more modifiednucleosides, 7 or more modified nucleosides, 8 or more modifiednucleosides, 9 or more modified nucleosides, 10 or more modifiednucleosides, 11 or more modified nucleosides, 12 or more modifiednucleosides, 13 or more modified nucleosides, 14 or more modifiednucleosides, 15 or more modified nucleosides, 16 or more modifiednucleosides, 17 or more modified nucleosides, 18 or more modifiednucleosides, 19 or more modified nucleosides, 20 or more modifiednucleosides, or 21 or more modified nucleosides.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesa lipid attached at a 3′ or 5′ terminus of the oligonucleotide. In someembodiments, the lipid comprises cholesterol, myristoyl, palmitoyl,stearoyl, lithocholoyl, docosanoyl, docosahexaenoyl, myristyl, palmitylstearyl, or α-tocopherol, or a combination thereof.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target mRNA, wherein the oligonucleotidecomprises a sugar moiety. The sugar moiety may include an N-acetylgalactose moiety (e.g. an N-acetylgalactosamine (GalNAc) moiety), anN-acetyl glucose moiety (e.g. an N-acetylglucosamine (GlcNAc) moiety), afucose moiety, or a mannose moiety. The sugar moiety may include 1, 2,3, or more sugar molecules. The sugar moiety may be attached at a 3′ or5′ terminus of the oligonucleotide. The sugar moiety may include anN-acetyl galactose moiety. The sugar moiety may include anN-acetylgalactosamine (GalNAc) moiety. The sugar moiety may include anN-acetyl glucose moiety. The sugar moiety may includeN-acetylglucosamine (GlcNAc) moiety. The sugar moiety may include afucose moiety. The sugar moiety may include a mannose moiety. N-acetylglucose. GlcNAc, fucose, or mannose may be useful for targetingmacrophages since they may target or bind a mannose receptor such asCD206.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target mRNA, wherein the oligonucleotidecomprises an N-acetylgalactosamine (GalNAc) moiety. GalNAc may be usefulfor hepatocyte targeting. In some embodiments, the composition comprisesGalNAc. In some embodiments, the composition comprises a GalNAcderivative. The GalNAc moiety may include 1, 2, 3, or more GalNAcmolecules. The GalNAc moiety may include a bivalent or trivalentbranched linker. The oligo may be attached to 1, 2 or 3 GalNAcs througha bivalent or trivalent branched linker. The GalNAc moiety may beattached at a 3′ or 5′ terminus of the oligonucleotide.

The oligonucleotide may include purines. Examples of purines includeadenine (A) or guanine (G), or modified versions thereof. Theoligonucleotide may include pyrimidines. Examples of pyrimidines includecytosine (C), thymine (T), or uracil (U), or modified versions thereof.

In some embodiments, purines of the oligonucleotide comprise 2′ fluoromodified purines. In some embodiments, purines of the oligonucleotidecomprise 2′-O-methyl modified purines. In some embodiments, purines ofthe oligonucleotide comprise a mixture of 2′ fluoro and 2′-O-methylmodified purines. In some embodiments, all purines of theoligonucleotide comprise 2′ fluoro modified purines. In someembodiments, all purines of the oligonucleotide comprise 2′-O-methylmodified purines. In some embodiments, all purines of theoligonucleotide comprise a mixture of 2′ fluoro and 2′-O-methyl modifiedpurines. In some embodiments, 2′-O-methyl includes 2′ O-methyl.

In some embodiments, pyrimidines of the oligonucleotide comprise 2′fluoro modified pyrimidines. In some embodiments, pyrimidines of theoligonucleotide comprise 2′-O-methyl modified pyrimidines. In someembodiments, pyrimidines of the oligonucleotide comprise a mixture of 2′fluoro and 2′-O-methyl modified pyrimidines. In some embodiments, allpyrimidines of the oligonucleotide comprise 2′ fluoro modifiedpyrimidines. In some embodiments, all pyrimidines of the oligonucleotidecomprise 2′-O-methyl modified pyrimidines. In some embodiments, allpyrimidines of the oligonucleotide comprise a mixture of 2′ fluoro and2′-O-methyl modified pyrimidines.

In some embodiments, purines of the oligonucleotide comprise 2′ fluoromodified purines, and pyrimidines of the oligonucleotide comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, purines of the oligonucleotide comprise 2′-O-methylmodified purines, and pyrimidines of the oligonucleotide comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, purines of the oligonucleotide comprise 2′ fluoro modifiedpurines, and pyrimidines of the oligonucleotide comprise 2′-O-methylmodified pyrimidines. In some embodiments, purines of theoligonucleotide comprise 2′-O-methyl modified purines, and pyrimidinesof the oligonucleotide comprise 2′ fluoro modified pyrimidines. In someembodiments, pyrimidines of the oligonucleotide comprise 2′ fluoromodified pyrimidines, and purines of the oligonucleotide comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, pyrimidines of the oligonucleotide comprise 2′-O-methylmodified pyrimidines, and purines of the oligonucleotide comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, pyrimidines of the oligonucleotide comprise 2′ fluoromodified pyrimidines, and purines of the oligonucleotide comprise2′-O-methyl modified purines. In some embodiments, pyrimidines of theoligonucleotide comprise 2′-O-methyl modified pyrimidines, and purinesof the oligonucleotide comprise 2′ fluoro modified purines.

In some embodiments, all purines of the oligonucleotide comprise 2′fluoro modified purines, and all pyrimidines of the oligonucleotidecomprise a mixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. Insome embodiments, all purines of the oligonucleotide comprise2′-O-methyl modified purines, and all pyrimidines of the oligonucleotidecomprise a mixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. Insome embodiments, all purines of the oligonucleotide comprise 2′ fluoromodified purines, and all pyrimidines of the oligonucleotide comprise2′-O-methyl modified pyrimidines. In some embodiments, all purines ofthe oligonucleotide comprise 2′-O-methyl modified purines, and allpyrimidines of the oligonucleotide comprise 2′ fluoro modifiedpyrimidines. In some embodiments, all pyrimidines of the oligonucleotidecomprise 2′ fluoro modified pyrimidines, and all purifies of theoligonucleotide comprise a mixture of 2′ fluoro and 2′-O-methyl modifiedpurines. In some embodiments, all pyrimidines of the oligonucleotidecomprise 2′-O-methyl modified pyrimidines, and all purines of theoligonucleotide comprise a mixture of 2′ fluoro and 2′-O-methyl modifiedpurines. In some embodiments, all pyrimidines of the oligonucleotidecomprise 2′ fluoro modified pyrimidines, and all purines of theoligonucleotide comprise 2′-O-methyl modified purines. In someembodiments, all pyrimidines of the oligonucleotide comprise 2′-O-methylmodified pyrimidines, and all purines of the oligonucleotide comprise 2′fluoro modified purifies.

2. siRNA Modification Patterns

In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, or 29 modified internucleoside linkages, or a range of modifiedinternucleoside linkages defined by any two of the aforementionedintegers. In some embodiments, the sense strand comprises 1-11 modifiedinternucleoside linkages. In some embodiments, the sense strandcomprises 2-6 modified internucleoside linkages. In some embodiments,the sense strand comprises 5 modified internucleoside linkages. In someembodiments, the sense strand comprises 4 modified internucleosidelinkages.

In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, or 29 modified internucleoside linkages, or a range ofmodified internucleoside linkages defined by any two of theaforementioned integers. In some embodiments, the antisense strandcomprises 1-11 modified internucleoside linkages. In some embodiments,the antisense strand comprises 2-6 modified internucleoside linkages. Insome embodiments, the antisense strand comprises 5 modifiedinternucleoside linkages. In some embodiments, the antisense strandcomprises 4 modified internucleoside linkages.

In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 modified nucleosides, or a range of modifiednucleosides defined by any two of the aforementioned integers. In someembodiments, the sense strand comprises 12-19 modified nucleosides. Insome embodiments, the sense strand comprises 12-21 modified nucleosides.In some embodiments, the sense strand comprises 19 modified nucleosides.In some embodiments, the sense strand comprises 21 modified nucleosides.

In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 modified nucleosides, or a range of modifiednucleosides defined by any two of the aforementioned integers. In someembodiments, the antisense strand comprises 12-19 modified nucleosides.In some embodiments, the antisense strand comprises 12-21 modifiednucleosides. In some embodiments, the antisense strand comprises 19modified nucleosides. In some embodiments, the antisense strandcomprises 21 modified nucleosides.

In some embodiments, the sense strand or the antisense strand furthercomprises at least 2 additional nucleosides attached to a 3′ terminus ofthe sense strand or the antisense strand. In some embodiments, the sensestrand or the antisense strand comprises 2 additional nucleosidesattached to a 3′ terminus of the sense strand or the antisense strand.As part of the sense strand, the additional nucleosides may or may notbe complementary to a target mRNA. The additional nucleosides of theantisense strand may include a uracil. The 2 additional nucleosides ofthe antisense strand may both include uracil.

In some embodiments, the sense strand or the sense strand furthercomprises at least 2 additional nucleosides attached to a 3′ terminus ofthe sense strand or the sense strand. In some embodiments, the sensestrand or the sense strand comprises 2 additional nucleosides attachedto a 3′ terminus of the sense strand or the sense strand. The additionalnucleosides of the sense strand may include a uracil. The 2 additionalnucleosides of the sense strand may both include uracil.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesan siRNA comprising a sense strand and an antisense strand, wherein thesense strand comprises a modification pattern. In some embodiments, thesense strand comprises modification pattern 1S:5′-NfsnsNfnNfnNfNfNfnNfnNfnNfnNfnNfsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, the sensestrand comprises modification pattern 1S #2:5′-NfnNfnNfnNfNfNfnNfnNfnNfnNfnNfsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, the sensestrand comprises modification pattern 2S:5′-nsnsnnNfnNfNfNfnnnnnnnnnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, the sensestrand comprises modification pattern 2S #2:5′-nnnnNfnNfNfNfnnnnnnnnnnsnsn-3′, wherein “Nf” is a 2′ fluoro-modifiednucleoside, “n” is a 2′ O-methyl modified nucleoside, and “s” is aphosphorothioate linkage. In some embodiments, the sense strandcomprises modification pattern 3S: 5′-nsnsnnNfnNfnNfnnnnnnnnnnsnsn-3′,wherein “Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methylmodified nucleoside, and “s” is a phosphorothioate linkage. In someembodiments, the sense strand comprises modification pattern 3S #2:5′-nnnnNfnNfnNfinnnnnnnnnnsnsn-3′, wherein “Nf” is a 2′ fluoro-modifiednucleoside, “n” is a 2′ O-methyl modified nucleoside, and “s” is aphosphorothioate linkage. In some embodiments, the sense strandcomprises modification pattern 4S:5′-NfsnsNfnNfnNfNfNfnNfnNfnNfnNfnNfsnsnN-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,“s” is a phosphorothioate linkage, and N comprises one or morenucleosides. In some embodiments, the sense strand comprisesmodification pattern 4S #2: 5′-NfnNfnNfnNfNfNfnNfnNfnNfnNfnNfsnsnN-3′,wherein “Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methylmodified nucleoside, “s” is a phosphorothioate linkage, and N comprisesone or more nucleosides. In some embodiments, the sense strand comprisesmodification pattern 5S: 5′-nsnsnnNfnNfNfNfnnnnnnnnnnsnsnN-3′, wherein“Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methyl modifiednucleoside, “s” is a phosphorothioate linkage, and N comprises one ormore nucleosides. In some embodiments, the sense strand comprisesmodification pattern 5S #2: 5′-nnnnNfnNfNfNfnnnnnnnnnnsnsnN-3′, wherein“Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methyl modifiednucleoside, “s” is a phosphorothioate linkage, and N comprises one ormore nucleosides. In some embodiments, the sense strand comprisesmodification pattern 6S: 5′-NfsnsNfnNfinNfnNfnNfnNfnNfnNfnNfsnsn-3′,wherein “Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methylmodified nucleoside, “s” is a phosphorothioate linkage, and N comprisesone or more nucleosides. In some embodiments, the sense strand comprisesmodification pattern 6S #2: 5′-NfnNfnNfnnNfniNfnNfnNfnNfnNfsnsn-3′,wherein “Nf” is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methylmodified nucleoside, “s” is a phosphorothioate linkage, and N comprisesone or more nucleosides. In some embodiments, the sense strand comprisesany one of modification patterns 1S, 2S, 3S, 4S, 5S, or 6S. In someembodiments, the sense strand comprises any one of modification patterns1S #2, 2S #2, 3S #2, 4S #2, 5S #2, or 6S #2. In some embodiments, thesense strand comprises any one of modification patterns 1S, 3S, 4S, or6S. In some embodiments, the sense strand comprises any one ofmodification patterns 1S #2, 3S #2, 4S #2, or 6S #2. Any one ofmodification patterns 1S-6S may include a GalNAc ligand attached to the3′ end. Any one of modification patterns 1 S-6S may include a GalNAcligand attached to the 5′ end. Any one of modification patterns 1S-6S #2may include a GalNAc ligand attached to the 3′ end. Any one ofmodification patterns 1S-6S #2 may include a GalNAc ligand attached tothe 5′ end.

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesan siRNA comprising a sense strand and an antisense strand, wherein theantisense strand comprises a modification pattern. In some embodiments,the antisense strand comprises modification pattern 1AS:5′-nsNfsnNfnNfnNfnNfnnnNfnNfnNfnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 2AS:5′-nsNfsnnnNfnNfNfnnnnNfnNfinnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 3AS:5′-nsNfsnnnNfnnnnnnnNfnNfnnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 4AS:5′-nsNfsnNfnNfnnnnnnnNfnNfnnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 5AS:5′-nsNfsnnnNfnNfnnnnnNfnNfnnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 6AS:5′-nsNfsnNfnNfnNfnNfnNfnNfnNfnNfnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 7AS:5′-nsNfsnNfnNfnNfNfnnnnNfnNfinnnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. In some embodiments, theantisense strand comprises modification pattern 8AS:5′-nsNfsnnnnnnnnnnnNfnnnnnsnsn-3′, wherein “Nf” is a 2′ fluoro-modifiednucleoside, “n” is a 2′ O-methyl modified nucleoside, and “s” is aphosphorothioate linkage. In some embodiments, the antisense strandcomprises modification pattern 9AS:5′-nsNfsnnnNfnnnnnnnNfnNfnNfnsnsn-3′, wherein “Nf” is a 2′fluoro-modified nucleoside, “n” is a 2′ O-methyl modified nucleoside,and “s” is a phosphorothioate linkage. Any one of modification patterns1AS-9AS may include a GalNAc ligand attached to the 3′ end. Any one ofmodification patterns 1AS-9AS may include a GalNAc ligand attached tothe 5′ end.

The modifications in any of the modification patterns may be optional.For example, 1, 2, 3, or more phosphorothioate linkages in any ofmodification patterns 1S-6S, 1S #2-6S #2, or 1AS-9AS may be replacedwith an unmodified linkage, or with a different modified linkage. Insome cases, 1, 2, 3, or more modified nucleosides in any of modificationpatterns 1S-6S or 1AS-9AS may be replaced with an unmodified nucleoside,or with a different modified nucleoside.

In some embodiments, the composition comprises an oligonucleotide thatinhibits the expression of a target mRNA wherein the oligonucleotidecomprises an siRNA comprising a sense strand and an antisense strand,wherein the sense strand comprises modification pattern 1S and theantisense strand comprises modification pattern 1AS, 2AS, 3AS, 4AS, 5AS,6AS, 7AS, 8AS, or 9AS. In some embodiments, the sense strand comprisesmodification pattern 2S and the antisense strand comprises modificationpattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In someembodiments, the sense strand comprises modification pattern 3S and theantisense strand comprises modification pattern 1AS, 2AS, 3AS, 4AS, 5AS,6AS, 7AS, 8AS, or 9AS. In some embodiments, the sense strand comprisesmodification pattern 4S and the antisense strand comprises modificationpattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In someembodiments, the sense strand comprises modification pattern 5S and theantisense strand comprises modification pattern 1AS, 2AS, 3AS, 4AS, 5AS,6AS, 7AS, 8AS, or 9AS. In some embodiments, the sense strand comprisesmodification pattern 6S and the antisense strand comprises modificationpattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In someembodiments, the sense strand comprises modification pattern 1AS, 2AS,3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In some embodiments, the sensestrand comprises modification pattern 1S #2 and the antisense strandcomprises modification pattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS,or 9AS. In some embodiments, the sense strand comprises modificationpattern 2S #2 and the antisense strand comprises modification pattern1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In some embodiments, thesense strand comprises modification pattern 3S #2 and the antisensestrand comprises modification pattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS,8AS, or 9AS. In some embodiments, the sense strand comprisesmodification pattern 4S #2 and the antisense strand comprisesmodification pattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. Insome embodiments, the sense strand comprises modification pattern 5S #2and the antisense strand comprises modification pattern 1 AS, 2AS, 3AS,4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In some embodiments, the sense strandcomprises modification pattern 6S #2 and the antisense strand comprisesmodification pattern 1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. Insome embodiments, the antisense strand comprises modification pattern1AS, 2AS, 3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS. In some embodiments, thesense strand or the antisense strand comprises modification patternASO1.

Any combination of sense and antisense modification patterns may beused. In some embodiments, the sense strand comprises modificationpattern 1S, and the antisense strand comprises modification pattern 1AS.In some embodiments, the sense strand comprises modification pattern 2S,and the antisense strand comprises modification pattern 2AS. In someembodiments, the sense strand comprises modification pattern 2S, and theantisense strand comprises modification pattern 3AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 1AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 4AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 5AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 6AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 7AS. In someembodiments, the sense strand comprises modification pattern 3S, and theantisense strand comprises modification pattern 8AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 1AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 4AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 5AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 6AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 7AS. In someembodiments, the sense strand comprises modification pattern 6S, and theantisense strand comprises modification pattern 8AS. In someembodiments, the sense strand comprises modification pattern 1S #2, andthe antisense strand comprises modification pattern 1 AS. In someembodiments, the sense strand comprises modification pattern 2S #2, andthe antisense strand comprises modification pattern 2AS. In someembodiments, the sense strand comprises modification pattern 2S #2, andthe antisense strand comprises modification pattern 3AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 1AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 4AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 5AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 6AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 7AS. In someembodiments, the sense strand comprises modification pattern 3S #2, andthe antisense strand comprises modification pattern 8AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 1AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 4AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 5AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 6AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 7AS. In someembodiments, the sense strand comprises modification pattern 6S #2, andthe antisense strand comprises modification pattern 8AS.

In some embodiments, purines of the sense strand comprise 2′ fluoromodified purines. In some embodiments, purines of the sense strandcomprise 2′-O-methyl modified purines. In some embodiments, purines ofthe sense strand comprise a mixture of 2′ fluoro and 2′-O-methylmodified purines. In some embodiments, all purines of the sense strandcomprise 2′ fluoro modified purines. In some embodiments, all purines ofthe sense strand comprise 2′-O-methyl modified purines. In someembodiments, all purines of the sense strand comprise a mixture of 2′fluoro and 2′-O-methyl modified purines.

In some embodiments, pyrimidines of the sense strand comprise 2′ fluoromodified pyrimidines. In some embodiments, pyrimidines of the sensestrand comprise 2′-O-methyl modified pyrimidines. In some embodiments,pyrimidines of the sense strand comprise a mixture of 2′ fluoro and2′-O-methyl modified pyrimidines. In some embodiments, all pyrimidinesof the sense strand comprise 2′ fluoro modified pyrimidines. In someembodiments, all pyrimidines of the sense strand comprise 2′-O-methylmodified pyrimidines. In some embodiments, all pyrimidines of the sensestrand comprise a mixture of 2′ fluoro and 2′-O-methyl modifiedpyrimidines.

In some embodiments, purines of the sense strand comprise 2′ fluoromodified purines, and pyrimidines of the sense strand comprise a mixtureof 2′ fluoro and 2′-O-methyl modified pyrimidines. In some embodiments,purines of the sense strand comprise 2′-O-methyl modified purines, andpyrimidines of the sense strand comprise a mixture of 2′ fluoro and2′-O-methyl modified pyrimidines. In some embodiments, purines of thesense strand comprise 2′ fluoro modified purines, and pyrimidines of thesense strand comprise 2′-O-methyl modified pyrimidines. In someembodiments, purines of the sense strand comprise 2′-O-methyl modifiedpurines, and pyrimidines of the sense strand comprise 2′ fluoro modifiedpyrimidines. In some embodiments, pyrimidines of the sense strandcomprise 2′ fluoro modified pyrimidines, and purines of the sense strandcomprise a mixture of 2′ fluoro and 2′-O-methyl modified purines. Insome embodiments, pyrimidines of the sense strand comprise 2′-O-methylmodified pyrimidines, and purines of the sense strand comprise a mixtureof 2′ fluoro and 2′-O-methyl modified purines. In some embodiments,pyrimidines of the sense strand comprise 2′ fluoro modified pyrimidines,and purines of the sense strand comprise 2′-O-methyl modified purines.In some embodiments, pyrimidines of the sense strand comprise2′-O-methyl modified pyrimidines, and purines of the sense strandcomprise 2′ fluoro modified purines.

In some embodiments, all purines of the sense strand comprise 2′ fluoromodified purines, and all pyrimidines of the sense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, all purines of the sense strand comprise 2′-O-methylmodified purines, and all pyrimidines of the sense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, all purines of the sense strand comprise 2′ fluoro modifiedpurines, and all pyrimidines of the sense strand comprise 2′-O-methylmodified pyrimidines. In some embodiments, all purines of the sensestrand comprise 2′-O-methyl modified purines, and all pyrimidines of thesense strand comprise 2′ fluoro modified pyrimidines. In someembodiments, all pyrimidines of the sense strand comprise 2′ fluoromodified pyrimidines, and all purines of the sense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, all pyrimidines of the sense strand comprise 2′-O-methylmodified pyrimidines, and all purines of the sense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, all pyrimidines of the sense strand comprise 2′ fluoromodified pyrimidines, and all purines of the sense strand comprise2′-O-methyl modified purines. In some embodiments, all pyrimidines ofthe sense strand comprise 2′-O-methyl modified pyrimidines, and allpurines of the sense strand comprise 2′ fluoro modified purines.

In some embodiments, purines of the antisense strand comprise 2′ fluoromodified purines. In some embodiments, purines of the antisense strandcomprise 2′-O-methyl modified purines. In some embodiments, purines ofthe antisense strand comprise a mixture of 2′ fluoro and 2′-O-methylmodified purines. In some embodiments, all purines of the antisensestrand comprise 2′ fluoro modified purines. In some embodiments, allpurines of the antisense strand comprise 2′-O-methyl modified purines.In some embodiments, all purines of the antisense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified purines.

In some embodiments, pyrimidines of the antisense strand comprise 2′fluoro modified pyrimidines. In some embodiments, pyrimidines of theantisense strand comprise 2′-O-methyl modified pyrimidines. In someembodiments, pyrimidines of the antisense strand comprise a mixture of2′ fluoro and 2′-O-methyl modified pyrimidines. In some embodiments, allpyrimidines of the antisense strand comprise 2′ fluoro modifiedpyrimidines. In some embodiments, all pyrimidines of the antisensestrand comprise 2′-O-methyl modified pyrimidines. In some embodiments,all pyrimidines of the antisense strand comprise a mixture of 2′ fluoroand 2′-O-methyl modified pyrimidines.

In some embodiments, purines of the antisense strand comprise 2′ fluoromodified purines, and pyrimidines of the antisense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, purines of the antisense strand comprise 2′-O-methylmodified purines, and pyrimidines of the antisense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. In someembodiments, purines of the antisense strand comprise 2′ fluoro modifiedpurines, and pyrimidines of the antisense strand comprise 2′-O-methylmodified pyrimidines. In some embodiments, purines of the antisensestrand comprise 2′-O-methyl modified purines, and pyrimidines of theantisense strand comprise 2′ fluoro modified pyrimidines. In someembodiments, pyrimidines of the antisense strand comprise 2′ fluoromodified pyrimidines, and purines of the antisense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, pyrimidines of the antisense strand comprise 2′-O-methylmodified pyrimidines, and purines of the antisense strand comprise amixture of 2′ fluoro and 2′-O-methyl modified purines. In someembodiments, pyrimidines of the antisense strand comprise 2′ fluoromodified pyrimidines, and purines of the antisense strand comprise2′-O-methyl modified purines. In some embodiments, pyrimidines of theantisense strand comprise 2′-O-methyl modified pyrimidines, and purinesof the antisense strand comprise 2′ fluoro modified purines.

In some embodiments, all purines of the antisense strand comprise 2′fluoro modified purines, and all pyrimidines of the antisense strandcomprise a mixture of 2′ fluoro and 2′-O-methyl modified pyrimidines. Insome embodiments, all purines of the antisense strand comprise2′-O-methyl modified purines, and all pyrimidines of the antisensestrand comprise a mixture of 2′ fluoro and 2′-O-methyl modifiedpyrimidines. In some embodiments, all purines of the antisense strandcomprise 2′ fluoro modified purines, and all pyrimidines of theantisense strand comprise 2′-O-methyl modified pyrimidines. In someembodiments, all purines of the antisense strand comprise 2′-O-methylmodified purines, and all pyrimidines of the antisense strand comprise2′ fluoro modified pyrimidines. In some embodiments, all pyrimidines ofthe antisense strand comprise 2′ fluoro modified pyrimidines, and allpurines of the antisense strand comprise a mixture of 2′ fluoro and2′-O-methyl modified purines. In some embodiments, all pyrimidines ofthe antisense strand comprise 2′-O-methyl modified pyrimidines, and allpurines of the antisense strand comprise a mixture of 2′ fluoro and2′-O-methyl modified purines. In some embodiments, all pyrimidines ofthe antisense strand comprise 2′ fluoro modified pyrimidines, and allpurines of the antisense strand comprise 2′-O-methyl modified purines.In some embodiments, all pyrimidines of the antisense strand comprise2′-O-methyl modified pyrimidines, and all purines of the antisensestrand comprise 2′ fluoro modified purines.

In some cases, the oligonucleotide comprises a particular modificationpattern. In some embodiments, position 9 counting from the 5′ end of theof a strand of the oligonucleotide may have a 2′F modification. In someembodiments, when position 9 of a strand of the oligonucleotide is apyrimidine, then all purines in a strand of the oligonucleotide have a2′OMe modification. In some embodiments, when position 9 is the onlypyrimidine between positions 5 and 11 of the sense stand, then position9 is the only position with a 2′F modification in a strand of theoligonucleotide. In some embodiments, when position 9 and only one otherbase between positions 5 and 11 of a strand of the oligonucleotide arepyrimidines, then both of these pyrimidines are the only two positionswith a 2′F modification in a strand of the oligonucleotide. In someembodiments, when position 9 and only two other bases between positions5 and 11 of a strand of the oligonucleotide are pyrimidines, and thosetwo other pyrimidines are in adjacent positions so that there would benot three 2′F modifications in a row, then any combination of 2′Fmodifications can be made that give three 2′F modifications in total. Insome embodiments, when there are more than 2 pyrimidines betweenpositions 5 and 11 of a strand of the oligonucleotide, then allcombinations of pyrimidines having the 2′F modification are allowed thathave three to five 2′F modifications in total, provided that a strand ofthe oligonucleotide does not have three 2′F modifications in a row. Insome cases, a strand of the oligonucleotide of any of the siRNAscomprises a modification pattern which conforms to any or all of these astrand of the oligonucleotide rules.

In some embodiments, when position 9 of a strand of the oligonucleotideis a purine, then all purines in a strand of the oligonucleotide have a2′OMe modification. In some embodiments, when position 9 is the onlypurine between positions 5 and 11 of the sense stand, then position 9 isthe only position with a 2′F modification in a strand of theoligonucleotide. In some embodiments, when position 9 and only one otherbase between positions 5 and 11 of a strand of the oligonucleotide arepurines, then both of these purines are the only two positions with a2′F modification in a strand of the oligonucleotide. In someembodiments, when position 9 and only two other bases between positions5 and 11 of a strand of the oligonucleotide are purines, and those twoother purines are in adjacent positions so that there would be not three2′F modifications in a row, then any combination of 2′F modificationscan be made that give three 2′F modifications in total. In someembodiments, when there are more than 2 purines between positions 5 and11 of a strand of the oligonucleotide, then all combinations of purineshaving the 2′F modification are allowed that have three to five 2′Fmodifications in total, provided that a strand of the oligonucleotidedoes not have three 2′F modifications in a row. In some cases, a strandof the oligonucleotide of any of the siRNAs comprises a modificationpattern which conforms to any or all of these a strand of theoligonucleotide rules.

In some cases, position 9 of a strand of the oligonucleotide can be a2′deoxy. In these cases, 2′F and 2′OMe modifications may occur at theother positions of a strand of the oligonucleotide. In some cases, astrand of the oligonucleotide of any of the siRNAs comprises amodification pattern which conforms to these a strand of theoligonucleotide rules.

In some embodiments, position nine of the sense strand comprises a 2′fluoro-modified pyrimidine. In some embodiments, all purines of thesense strand comprise 2′-O-methyl modified purines. In some embodiments,1, 2, 3, 4, or 5 pyrimidines between positions 5 and 11 comprise a2′flouro-modified pyrimidine, provided there are not three 2′fluoro-modified pyrimidines in a row. In some embodiments, theodd-numbered positions of the antisense strand comprise 2′-O-methylmodified nucleotides. In some embodiments, the even-numbered positionsof the antisense strand comprise 2′flouro-modified nucleotides andunmodified deoxyribonucleotide. In some embodiments, the even-numberedpositions of the antisense strand comprise 2′flouro-modifiednucleotides, 2′-O-methyl modified nucleotides and unmodifieddeoxyribonucleotide. In some embodiments, position nine of the sensestrand comprises a 2′ fluoro-modified pyrimidine; all purines of thesense strand comprises 2′-O-methyl modified purines; 1, 2, 3, 4, or 5pyrimidines between positions 5 and 11 comprise a 2′flouro-modifiedpyrimidine, provided there are not three 2′ fluoro-modified pyrimidinesin a row; the odd-numbered positions of the antisense strand comprise2′-O-methyl modified nucleotides; and the even-numbered positions of theantisense strand comprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotides.

In some embodiments, position nine of the sense strand comprises a 2′fluoro-modified purine. In some embodiments, all pyrimidines of thesense strand comprise 2′-O-methyl modified purines. In some embodiments,1, 2, 3, 4, or 5 purines between positions 5 and 11 comprise a2′flouro-modified purine, provided there are not three 2′fluoro-modified purine in a row. In some embodiments, the odd-numberedpositions of the antisense strand comprise 2′-O-methyl modifiednucleotides. In some embodiments, the even-numbered positions of theantisense strand comprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotide. In some embodiments, the even-numbered positions ofthe antisense strand comprise 2′flouro-modified nucleotides, 2′-O-methylmodified nucleotides and unmodified deoxyribonucleotide. In someembodiments, position nine of the sense strand comprises a 2′fluoro-modified purine; all pyrimidine of the sense strand comprises2′-O-methyl modified pyrimidines; 1, 2, 3, 4, or 5 purines betweenpositions 5 and 11 comprise a 2′flouro-modified purines, provided thereare not three 2′ fluoro-modified purines in a row; the odd-numberedpositions of the antisense strand comprise 2′-O-methyl modifiednucleotides; and the even-numbered positions of the antisense strandcomprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotides. In some embodiments, there are not three 2′fluoro-modified purines in a row. In some embodiments, there are notthree 2′ fluoro-modified pyrimidines in a row.

In some embodiments, position nine of the sense strand comprises anunmodified deoxyribonucleotide. In some embodiments, positions 5, 7, and8 of the sense strand comprise 2′fluoro-modified nucleotides. In someembodiments, all pyrimidines in positions 10 to 21 of the sense strandcomprise 2′-O-methyl modified pyrimidines and all purines in positions10 to 21 of the comprise 2′-O-methyl modified purines or2′fluoro-modified purines. In some embodiments, the odd-numberedpositions of the antisense strand comprise 2′-O-methyl modifiednucleotides. In some embodiments, the even-numbered positions of theantisense strand comprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotides. In some embodiments, the even-numbered positionsof the antisense strand comprise 2′flouro-modified nucleotides,2′-O-methyl modified nucleotides and unmodified deoxyribonucleotides. Insome embodiments, position nine of the sense strand comprises anunmodified deoxyribonucleotide; positions 5, 7, and 8 of the sensestrand comprise 2′fluoro-modified nucleotides; all pyrimidines inpositions 10 to 21 of the sense strand comprise 2′-O-methyl modifiedpyrimidines and all purines in positions 10 to 21 of the comprise2′-O-methyl modified purines or 2′fluoro-modified purines; theodd-numbered positions of the antisense strand comprise 2′-O-methylmodified nucleotides; and the even-numbered positions of the antisensestrand comprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotides.

In some embodiments, position nine of the sense strand comprises anunmodified deoxyribonucleotide. In some embodiments, positions 5, 7, and8 of the sense strand comprise 2′fluoro-modified nucleotides. In someembodiments, all purines in positions 10 to 21 of the sense strandcomprise 2′-O-methyl modified purines and all pyrimidines in positions10 to 21 of the comprise 2′-O-methyl modified pyrimidines or2′fluoro-modified pyrimidines. In some embodiments, the odd-numberedpositions of the antisense strand comprise 2′-O-methyl modifiednucleotides. In some embodiments, the even-numbered positions of theantisense strand comprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotides. In some embodiments, the even-numbered positionsof the antisense strand comprise 2′flouro-modified nucleotides,2′-O-methyl modified nucleotides and unmodified deoxyribonucleotides. Insome embodiments, position nine of the sense strand comprises anunmodified deoxyribonucleotide; positions 5, 7, and 8 of the sensestrand comprise 2′fluoro-modified nucleotides; all purines in positions10 to 21 of the sense strand comprise 2′-O-methyl modified purines andall pyrimidines in positions 10 to 21 of the comprise 2′-O-methylmodified pyrimidines or 2′fluoro-modified pyrimidines; the odd-numberedpositions of the antisense strand comprise 2′-O-methyl modifiednucleotides; and the even-numbered positions of the antisense strandcomprise 2′flouro-modified nucleotides and unmodifieddeoxyribonucleotide.

3. ASO Modification Patterns

In some embodiments, the composition comprises an oligonucleotide thatbinds to a target oligonucleotide, wherein the oligonucleotide comprisesan antisense oligonucleotide (ASO). In some embodiments, the ASOcomprises modification pattern ASO1:5′-nsnsnsnsnsdNsdNsdNsdNsdNsdNsdNsdNsdNsdNsnsnsnsnsn-3′, wherein “dN” isany deoxynucleotide, “n” is a 2′O-methyl or 2′O-methoxyethyl-modifiednucleoside, and “s” is a phosphorothioate linkage. In some embodiments,the ASO comprises modification pattern 1S, 2S, 3S, 4S, 5S, 6S, 1AS, 2AS,3AS, 4AS, 5AS, 6AS, 7AS, 8AS, or 9AS.

In some embodiments, the ASO is conjugated to a GalNAc moiety. TheGalNAc moiety may be conjugated to the ASO at a 5′ terminus or the 3′terminus. In some embodiments, the GalNAc moiety is conjugated to the 5′terminus of the ASO. In some embodiments, the GalNAc moiety isconjugated to the 3′ terminus of the ASO.

C. Formulations

In some embodiments, the composition is a pharmaceutical composition. Insome embodiments, the composition is sterile. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier. Theformulation may include a compound such as a GalNAc moiety, and anoligonucleotide conjugated to a GalNAc moiety described herein.

In some embodiments, the pharmaceutically acceptable carrier compriseswater. In some embodiments, the pharmaceutically acceptable carriercomprises a buffer. In some embodiments, the pharmaceutically acceptablecarrier comprises a saline solution. In some embodiments, thepharmaceutically acceptable carrier comprises water, a buffer, or asaline solution. In some embodiments, the composition comprises aliposome. In some embodiments, the pharmaceutically acceptable carriercomprises liposomes, lipids, nanoparticles, proteins, protein-antibodycomplexes, peptides, cellulose, nanogel, or a combination thereof.

II. METHODS AND USES

Disclosed herein, in some embodiments, are methods of administering acomposition described herein to a subject. Some embodiments relate touse a composition described herein, such as administering thecomposition to a subject.

Some embodiments relate to a method of treating a disorder in a subjectin need thereof. Some embodiments relate to use of a compositiondescribed herein in the method of treatment. Some embodiments includeadministering a composition described herein to a subject with thedisorder. In some embodiments, the administration treats the disorder inthe subject. In some embodiments, the composition treats the disorder inthe subject.

In some embodiments, the treatment comprises prevention, inhibition, orreversion of the disorder in the subject. Some embodiments relate to useof a composition described herein in the method of preventing,inhibiting, or reversing the disorder. Some embodiments relate to amethod of preventing, inhibiting, or reversing a disorder a disorder ina subject in need thereof. Some embodiments include administering acomposition described herein to a subject with the disorder. In someembodiments, the administration prevents, inhibits, or reverses thedisorder in the subject. In some embodiments, the composition prevents,inhibits, or reverses the disorder in the subject.

Some embodiments relate to a method of preventing a disorder a disorderin a subject in need thereof. Some embodiments relate to use of acomposition described herein in the method of preventing the disorder.Some embodiments include administering a composition described herein toa subject with the disorder. In some embodiments, the administrationprevents the disorder in the subject. In some embodiments, thecomposition prevents the disorder in the subject.

Some embodiments relate to a method of inhibiting a disorder a disorderin a subject in need thereof. Some embodiments relate to use of acomposition described herein in the method of inhibiting the disorder.Some embodiments include administering a composition described herein toa subject with the disorder. In some embodiments, the administrationinhibits the disorder in the subject. In some embodiments, thecomposition inhibits the disorder in the subject.

Some embodiments relate to a method of reversing a disorder a disorderin a subject in need thereof. Some embodiments relate to use of acomposition described herein in the method of reversing the disorder.Some embodiments include administering a composition described herein toa subject with the disorder. In some embodiments, the administrationreverses the disorder in the subject. In some embodiments, thecomposition reverses the disorder in the subject.

A. Disorders

Some embodiments of the methods described herein include treating adisorder in a subject in need thereof. In some embodiments, the disorderis a liver disorder. Non-limiting examples of liver disorders includeliver inflammation, liver cancer, liver fibrosis, cholestasis, a gallbladder disease, a biliary tree disease, alcoholic liver disease,non-alcoholic steatohepatitis, a liver infection, or an inherited liverdisorder. In some embodiments, the liver disorder comprises liverinflammation. In some embodiments, the liver disorder comprises livercancer. In some embodiments, the liver disorder comprises liverfibrosis. In some embodiments, the liver disorder comprises cholestasis.In some embodiments, the liver disorder comprises a gall bladderdisease. In some embodiments, the liver disorder comprises a biliarytree disease. In some embodiments, the liver disorder comprisesalcoholic liver disease. In some embodiments, the liver disordercomprises non-alcoholic steatohepatitis.

In some embodiments, the liver disorder comprises a liver infection. Insome embodiments, the liver infection comprises hepatitis A. In someembodiments, the liver infection comprises hepatitis B. In someembodiments, the liver infection comprises hepatitis C.

In some embodiments, the liver disorder comprises an inherited liverdisorder. In some embodiments, the inherited liver disorder compriseshemochromatosis. In some embodiments, the inherited liver disordercomprises Wilson disease.

B. Subjects

Some embodiments of the methods described herein include treatment of asubject. Non-limiting examples of subjects include vertebrates, animals,mammals, dogs, cats, cattle, rodents, mice, rats, primates, monkeys, andhumans. In some embodiments, the subject is a vertebrate. In someembodiments, the subject is an animal. In some embodiments, the subjectis a mammal. In some embodiments, the subject is a dog. In someembodiments, the subject is a cat. In some embodiments, the subject is acattle. In some embodiments, the subject is a mouse. In someembodiments, the subject is a rat. In some embodiments, the subject is aprimate. In some embodiments, the subject is a monkey. In someembodiments, the subject is an animal, a mammal, a dog, a cat, cattle, arodent, a mouse, a rat, a primate, or a monkey. In some embodiments, thesubject is a human.

In some embodiments, the subject is male. In some embodiments, thesubject is female.

In some embodiments, the subject has a body mass index (BMI) of 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, or more, or a range defined by any two of theaforementioned integers. In some embodiments, the subject is overweight.In some embodiments, the subject has a BMI of 25 or more. In someembodiments, the subject has a BMI of 25-29. In some embodiments, thesubject is obese. In some embodiments, the subject has a BMI of 30 ormore. In some embodiments, the subject has a BMI of 30-39. In someembodiments, the subject has a BMI of 40-50. In some embodiments, thesubject has a BMI of 25-50.

In some embodiments, the subject is ≥90 years of age. In someembodiments, the subject is ≥85 years of age. In some embodiments, thesubject is ≥80 years of age. In some embodiments, the subject is ≥70years of age. In some embodiments, the subject is ≥60 years of age. Insome embodiments, the subject is ≥50 years of age. In some embodiments,the subject is ≥40 years of age. In some embodiments, the subject is ≥30years of age. In some embodiments, the subject is ≥20 years of age. Insome embodiments, the subject is ≥10 years of age. In some embodiments,the subject is ≥1 years of age. In some embodiments, the subject is ≥0years of age.

In some embodiments, the subject is ≤100 years of age. In someembodiments, the subject is ≤90 years of age. In some embodiments, thesubject is ≤85 years of age. In some embodiments, the subject is ≤80years of age. In some embodiments, the subject is ≤70 years of age. Insome embodiments, the subject is ≤60 years of age. In some embodiments,the subject is ≤50 years of age. In some embodiments, the subject is ≤40years of age. In some embodiments, the subject is ≤30 years of age. Insome embodiments, the subject is ≤20 years of age. In some embodiments,the subject is ≤10 years of age. In some embodiments, the subject is ≤1years of age.

In some embodiments, the subject is between 0 and 100 years of age. Insome embodiments, the subject is between 20 and 90 years of age. In someembodiments, the subject is between 30 and 80 years of age. In someembodiments, the subject is between 40 and 75 years of age. In someembodiments, the subject is between 50 and 70 years of age. In someembodiments, the subject is between 40 and 85 years of age.

C. Baseline Measurements

Some embodiments of the methods described herein include obtaining abaseline measurement from a subject. For example, in some embodiments, abaseline measurement is obtained from the subject prior to treating thesubject. Non-limiting examples of baseline measurements include abaseline symptom (e.g. a liver disorder symptom) measurement, a baselineprotective phenotype measurement, a baseline target oligonucleotide(e.g. mRNA) measurement or a baseline target protein measurement.

In some embodiments, the baseline measurement is obtained directly fromthe subject. In some embodiments, the baseline measurement is obtainedby observation, for example by observation of the subject or of thesubject's tissue. In some embodiments, the baseline measurement isobtained noninvasively using an imaging device. In some embodiments, thebaseline measurement is obtained in a sample from the subject. In someembodiments, the baseline measurement is obtained in one or morehistological tissue sections. In some embodiments, the baselinemeasurement is obtained by performing an assay such as an immunoassay, acolorimetric assay, or a fluorescence assay, on the sample obtained fromthe subject. In some embodiments, the baseline measurement is obtainedby an immunoassay, a colorimetric assay, or a fluorescence assay. Insome embodiments, the baseline measurement is obtained by PCR.

In some embodiments, the baseline measurement is a baseline symptommeasurement. The symptom may be a symptom of a disorder associated witha target oligonucleotide. The disorder may be a liver disorder.

In some embodiments, the baseline measurement is a baseline protectivephenotype measurement. The protective phenotype may protect a subjectfrom having a disorder associated with a target oligonucleotide. Theprotective phenotype may be inversely correlated with an incidence ofthe disorder.

In some embodiments, the baseline measurement is a baseline targetprotein measurement. In some embodiments, the baseline target proteinmeasurement comprises a baseline target protein level. In someembodiments, the baseline target protein level is indicated as a mass orpercentage of target protein per sample weight. In some embodiments, thebaseline target protein level is indicated as a mass or percentage oftarget protein per sample volume. In some embodiments, the baselinetarget protein level is indicated as a mass or percentage of targetprotein per total protein within the sample. In some embodiments, thebaseline target protein measurement is a baseline liver or hepatocytetarget protein measurement. In some embodiments, the baseline targetprotein measurement is a baseline circulating target proteinmeasurement. In some embodiments, the baseline target proteinmeasurement is obtained by an assay such as an immunoassay, acolorimetric assay, or a fluorescence assay.

In some embodiments, the baseline measurement is a baseline target mRNAmeasurement. In some embodiments, the baseline target mRNA measurementcomprises a baseline target mRNA level. In some embodiments, thebaseline target mRNA level is indicated as a mass or percentage oftarget mRNA per sample weight. In some embodiments, the baseline targetmRNA level is indicated as a mass or percentage of target mRNA persample volume. In some embodiments, the baseline target mRNA level isindicated as a mass or percentage of target mRNA per total mRNA withinthe sample. In some embodiments, the baseline target mRNA level isindicated as a mass or percentage of target mRNA per total nucleic acidswithin the sample. In some embodiments, the baseline target mRNA levelis indicated relative to another mRNA level, such as an mRNA level of ahousekeeping gene, within the sample. In some embodiments, the baselinetarget mRNA measurement is a baseline liver or hepatocyte target mRNAmeasurement. In some embodiments, the baseline target mRNA measurementis obtained by an assay such as a polymerase chain reaction (PCR) assay.In some embodiments, the PCR comprises quantitative PCR (qPCR). In someembodiments, the PCR comprises reverse transcription of the target mRNA.

Some embodiments of the methods described herein include obtaining asample from a subject. In some embodiments, the baseline measurement isobtained in a sample obtained from the subject. In some embodiments, thesample is obtained from the subject prior to administration or treatmentof the subject with a composition described herein. In some embodiments,a baseline measurement is obtained in a sample obtained from the subjectprior to administering the composition to the subject. In someembodiments, the sample is obtained from the subject in a fasted state.In some embodiments, the sample is obtained from the subject after anovernight fasting period. In some embodiments, the sample is obtainedfrom the subject in a fed state.

In some embodiments, the sample comprises a fluid. In some embodiments,the sample is a fluid sample. In some embodiments, the sample is ablood, plasma, or serum sample. In some embodiments, the samplecomprises blood. In some embodiments, the sample is a blood sample. Insome embodiments, the sample is a whole-blood sample. In someembodiments, the blood is fractionated or centrifuged. In someembodiments, the sample comprises plasma. In some embodiments, thesample is a plasma sample. In some embodiments, the sample comprisesserum. In some embodiments, the sample is a serum sample.

In some embodiments, the sample comprises a tissue. In some embodiments,the sample is a tissue sample. In some embodiments, the sample comprisesliver tissue. In some embodiments, the sample is a liver sample. In someembodiments, the sample comprises hepatocytes. In some embodiments, thesample consists of hepatocytes. For example, the baseline target mRNAmeasurement, or the baseline target protein measurement, may be obtainedin a liver or hepatocyte sample from the patient. In some embodiments,the sample comprises adipose tissue. In some embodiments, the sample isan adipose sample. The adipose sample may comprise or consist of whiteadipose tissue. The adipose sample may comprise or consist of brownadipose tissue. In some embodiments, the sample comprises kidney tissue.In some embodiments, the sample is an kidney sample. In someembodiments, the sample comprises cardiac tissue such as ventricular oratrial tissue. In some embodiments, the sample is a cardiac sample. Insome embodiments, the sample comprises intestinal tissue such as smallintestinal tissue. In some embodiments, the sample is a small intestinesample. In some embodiments, the sample comprises lymph node tissue suchas mesenteric lymph node tissue. In some embodiments, the sample is amesenteric lymph node sample. In some embodiments, the sample comprisesmuscle tissue. In some embodiments, the sample is an muscle sample. Insome embodiments, the tissue sample comprises liver, adipose, kidney, orcardiac tissue. In some embodiments, the tissue sample comprises brownadipose tissue, white adipose tissue, kidney tissue, intestinal tissue,mesenteric lymph node, or muscle tissue.

D. Effects

In some embodiments, the composition or administration of thecomposition affects a measurement such as symptom (e.g. a liver disordersymptom) measurement, a protective phenotype measurement, a targetoligonucleotide (e.g. mRNA) measurement or a target protein measurement(e.g. liver tissue target protein levels), relative to the baselinemeasurement.

Some embodiments of the methods described herein include obtaining themeasurement from a subject. For example, the measurement may be obtainedfrom the subject after treating the subject. In some embodiments, themeasurement is obtained in a second sample (such as a fluid or tissuesample described herein) obtained from the subject after the compositionis administered to the subject. In some embodiments, the measurement isan indication that the disorder has been treated.

In some embodiments, the measurement is obtained directly from thesubject. In some embodiments, the measurement is obtained noninvasivelyusing an imaging device. In some embodiments, the measurement isobtained in a second sample from the subject. In some embodiments, themeasurement is obtained in one or more histological tissue sections. Insome embodiments, the measurement is obtained by performing an assay onthe second sample obtained from the subject. In some embodiments, themeasurement is obtained by an assay, such as an assay described herein.In some embodiments, the assay is an immunoassay, a colorimetric assay,a fluorescence assay, or a PCR assay. In some embodiments, themeasurement is obtained by an assay such as an immunoassay, acolorimetric assay, or a fluorescence assay. In some embodiments, themeasurement is obtained by PCR. In some embodiments, the measurement isobtained by histology. In some embodiments, the measurement is obtainedby observation. In some embodiments, additional measurements are made,such as in a 3rd sample, a 4th sample, or a fifth sample.

In some embodiments, the measurement is obtained within 1 hour, within 2hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours,within 12 hours, within 18 hours, or within 24 hours after theadministration of the composition. In some embodiments, the measurementis obtained within 1 day, within 2 days, within 3 days, within 4 days,within 5 days, within 6 days, or within 7 days after the administrationof the composition. In some embodiments, the measurement is obtainedwithin 1 week, within 2 weeks, within 3 weeks, within 1 month, within 2months, within 3 months, within 6 months, within 1 year, within 2 years,within 3 years, within 4 years, or within 5 years after theadministration of the composition. In some embodiments, the measurementis obtained after 1 hour, after 2 hours, after 3 hours, after 4 hours,after 5 hours, after 6 hours, after 12 hours, after 18 hours, or after24 hours after the administration of the composition. In someembodiments, the measurement is obtained after 1 day, after 2 days,after 3 days, after 4 days, after 5 days, after 6 days, or after 7 daysafter the administration of the composition. In some embodiments, themeasurement is obtained after 1 week, after 2 weeks, after 3 weeks,after 1 month, after 2 months, after 3 months, after 6 months, after 1year, after 2 years, after 3 years, after 4 years, or after 5 years,following the administration of the composition.

In some embodiments, the measurement of the symptom or the parameterrelated to the disorder in the subject is decreased or affected for anextended period of time, relative to the baseline measurement. In someembodiments, the measurement is decreased or affected for at least about1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 7 days, about 8 days, about 9 days, about 10 days, about 11days, about 12 days, about 13 days, about 14 days, about 15 days, about16 days, about 17 days, about 18 days, about 19 days, about 20 days,about 21 days, about 22 days, about 23 days, about 24 days, about 25days, about 26 days, about 27 days, about 28 days, about 29 days, about30 days, about 35 days, about 40 days, about 45 days, about 50 days,about 55 days, about 60 days, about 65 days, about 70 days, about 75days, about 80 days, about 85 days, about 90 days, about 95 days, about100 days, about 105 days, about 110 days, about 115 days, or about 120days, following administration, or a range of time followingadministration comprising a range defined by any two of theaforementioned numbers of days. In some embodiments, the measurement isdecreased or affected for at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 14 days, at least 15days, at least 16 days, at least 17 days, at least 18 days, at least 19days, at least 20 days, at least 21 days, at least 22 days, at least 23days, at least 24 days, at least 25 days, at least 26 days, at least 27days, at least 28 days, at least 29 days, at least 30 days, at least 35days, at least 40 days, at least 45 days, at least 50 days, at least 55days, at least 60 days, at least 65 days, at least 70 days, at least 75days, at least 80 days, at least 85 days, at least 90 days, at least 95days, at least 100 days, at least 105 days, at least 110 days, at least115 days, or at least 120 days, following administration. In someembodiments, the measurement is decreased or affected for no more than 1day, no more than 2 days, no more than 3 days, no more than 4 days, nomore than 5 days, no more than 6 days, no more than 7 days, no more than8 days, no more than 9 days, no more than 10 days, no more than 11 days,no more than 12 days, no more than 13 days, no more than 14 days, nomore than 15 days, no more than 16 days, no more than 17 days, no morethan 18 days, no more than 19 days, no more than 20 days, no more than21 days, no more than 22 days, no more than 23 days, no more than 24days, no more than 25 days, no more than 26 days, no more than 27 days,no more than 28 days, no more than 29 days, no more than 30 days, nomore than 35 days, no more than 40 days, no more than 45 days, no morethan 50 days, no more than 55 days, no more than 60 days, no more than65 days, no more than 70 days, no more than 75 days, no more than 80days, no more than 85 days, no more than 90 days, no more than 95 days,no more than 100 days, no more than 105 days, no more than 110 days, nomore than 115 days, or no more than 120 days, following administration.In some embodiments, the measurement is decreased or affected for atleast about 5 days. In some embodiments, the measurement is decreased oraffected for at least about 10 days. In some embodiments, themeasurement is decreased or affected for at least about 15 days. In someembodiments, the measurement is decreased or affected for at least about20 days. In some embodiments, the measurement is decreased or affectedfor at least about 25 days. In some embodiments, the measurement isdecreased or affected for at least about 30 days. In some embodiments,the measurement is decreased or affected for at least about 40 days. Insome embodiments, the measurement is decreased or affected for at leastabout 50 days. In some embodiments, the measurement is decreased oraffected for at least about 60 days. In some embodiments, themeasurement is decreased or affected for at least about 70 days. In someembodiments, the measurement is decreased or affected for at least about80 days. In some embodiments, the measurement is decreased or affectedfor at least about 90 days. In some embodiments, the measurement isdecreased or affected for at least about 100 days. In some embodiments,the measurement is decreased or affected for at least about 110 days. Insome embodiments, the measurement is decreased or affected for at leastabout 120 days. An example of a measurement of a symptom or parameterrelated to a disorder may include a target mRNA measurement, a targetprotein measurement, a biomarker measurement, or a physiologicalmeasurement. The measurement may be in a tissue (e.g. liver) or in abiofluid (e.g. blood, serum, or plasma).

In some embodiments, the composition reduces the symptom measurementrelative to the baseline symptom measurement. In some embodiments, thereduction is measured in a second tissue sample obtained from thesubject after administering the composition to the subject. In someembodiments, the reduction is measured directly in the subject afteradministering the composition to the subject. In some embodiments, thesymptom measurement is decreased by about 2.5% or more, about 5% ormore, or about 7.5% or more, relative to the baseline symptommeasurement. In some embodiments, the symptom measurement is decreasedby about 10% or more, relative to the baseline symptom measurement. Insome embodiments, the symptom measurement is decreased by about 20% ormore, about 30% or more, about 40% or more, about 50% or more, about 60%or more, about 70% or more, about 80% or more, about 90% or more,relative to the baseline symptom measurement. In some embodiments, thesymptom measurement is decreased by no more than about 2.5%, no morethan about 5%, or no more than about 7.5%, relative to the baselinesymptom measurement. In some embodiments, the symptom measurement isdecreased by no more than about 10%, relative to the baseline symptommeasurement. In some embodiments, the symptom measurement is decreasedby no more than about 20%, no more than about 30%, no more than about40%, no more than about 50%, no more than about 60%, no more than about70%, no more than about 80%, no more than about 90%, or no more thanabout 100% relative to the baseline symptom measurement. In someembodiments, the symptom measurement is decreased by 2.5%, 5%, 7.5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or by a rangedefined by any of the two aforementioned percentages.

In some embodiments, the composition increases the protective phenotypemeasurement relative to the baseline protective phenotype measurement.In some embodiments, the increase is measured in a second tissue sampleobtained from the subject after administering the composition to thesubject. In some embodiments, the increase is measured directly in thesubject after administering the composition to the subject. In someembodiments, the protective phenotype measurement is increased by about2.5% or more, about 5% or more, or about 7.5% or more, relative to thebaseline protective phenotype measurement. In some embodiments, theprotective phenotype measurement is increased by about 10% or more,relative to the baseline protective phenotype measurement. In someembodiments, the protective phenotype measurement is increased by about20% or more, about 30% or more, about 40% or more, about 50% or more,about 60% or more, about 70% or more, about 80% or more, about 90% ormore, relative to the baseline protective phenotype measurement. In someembodiments, the protective phenotype measurement is increased by about100% or more, increased by about 250% or more, increased by about 500%or more, increased by about 750% or more, or increased by about 1000% ormore, relative to the baseline protective phenotype measurement. In someembodiments, the protective phenotype measurement is increased by nomore than about 2.5%, no more than about 5%, or no more than about 7.5%,relative to the baseline protective phenotype measurement. In someembodiments, the protective phenotype measurement is increased by nomore than about 10%, relative to the baseline protective phenotypemeasurement. In some embodiments, the protective phenotype measurementis increased by no more than about 20%, no more than about 30%, no morethan about 40%, no more than about 50%, no more than about 60%, no morethan about 70%, no more than about 80%, no more than about 90%, or nomore than about 100% relative to the baseline protective phenotypemeasurement. In some embodiments, the protective phenotype measurementis increased by no more than about 100%, increased by no more than about250%, increased by no more than about 500%, increased by no more thanabout 750%, or increased by no more than about 1000%, relative to thebaseline protective phenotype measurement. In some embodiments, theprotective phenotype measurement is increased by 2.5%, 5%, 7.5%, 100%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 250%, 500%, 750%, or1000%, or by a range defined by any of the two aforementionedpercentages.

In some embodiments, the measurement is a target protein measurement. Insome embodiments, the target protein measurement comprises a targetprotein level. In some embodiments, the target protein level isindicated as a mass or percentage of target protein per sample weight.In some embodiments, the target protein level is indicated as a mass orpercentage of target protein per sample volume. In some embodiments, thetarget protein level is indicated as a mass or percentage of targetprotein per total protein within the sample. In some embodiments, thetarget protein measurement is a cell (e.g. hepatocyte) target proteinmeasurement. In some embodiments, the target protein measurement is atissue (e.g. liver tissue) target protein measurement. In someembodiments, the target protein measurement is a circulating targetprotein measurement. In some embodiments, the baseline target proteinmeasurement is obtained by an assay such as an immunoassay, acolorimetric assay, or a fluorescence assay.

In some embodiments, the composition reduces the target proteinmeasurement relative to the baseline target protein measurement. In someembodiments, the composition reduces tissue target protein levels (suchas, but not limited to, liver tissue target protein levels) relative tothe baseline target protein measurement. In some embodiments, thecomposition reduces cell target protein levels (such as, but not limitedto, hepatocyte target protein levels) relative to the baseline targetprotein measurement. In some embodiments, the composition reducescirculating target protein levels relative to the baseline targetprotein measurement. In some embodiments, the reduced target proteinlevels are measured in a second sample obtained from the subject afteradministering the composition to the subject.

In some embodiments, the target protein measurement is decreased byabout 2.5% or more, about 5% or more, or about 7.5% or more, relative tothe baseline target protein measurement. In some embodiments, the targetprotein measurement is decreased by about 10% or more, relative to thebaseline target protein measurement. In some embodiments, the targetprotein measurement is decreased by about 20% or more, about 30% ormore, about 40% or more, about 50% or more, about 60% or more, about 70%or more, about 80% or more, about 90% or more, or about 100% relative tothe baseline target protein measurement. In some embodiments, the targetprotein measurement is decreased by no more than about 2.5%, no morethan about 5%, or no more than about 7.5%, relative to the baselinetarget protein measurement. In some embodiments, the target proteinmeasurement is decreased by no more than about 10%, relative to thebaseline target protein measurement. In some embodiments, the targetprotein measurement is decreased by no more than about 20%, no more thanabout 30%, no more than about 40%, no more than about 50%, no more thanabout 60%, no more than about 70%, no more than about 80%, no more thanabout 90%, or about 100% relative to the baseline target proteinmeasurement. In some embodiments, the target protein measurement isdecreased by 2.5%, 5%, 7.5%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, or by a range defined by any of the two aforementionedpercentages. The target protein measurement may be decreased for anextended period of time. In some embodiments, the target proteinmeasurement is decreased for 7 days, 14 days, 28 days, 42 days, 56 days,70 days, 77 days, 84 days, 91 days, 98 days, 105 days, or a rangetherebetween. In some embodiments, the target protein measurement isdecreased for about 7 days, about 14 days, about 28 days, about 42 days,about 56 days, about 70 days, about 77 days, about 84 days, about 91days, about 98 days, about 105 days, or a range therebetween.

In some embodiments, the measurement is a target mRNA measurement. Insome embodiments, the target mRNA measurement comprises a target mRNAlevel. In some embodiments, the target mRNA level is indicated as a massor percentage of target mRNA per sample weight. In some embodiments, thetarget mRNA level is indicated as a mass or percentage of target mRNAper sample volume. In some embodiments, the target mRNA level isindicated as a mass or percentage of target mRNA per total mRNA withinthe sample. In some embodiments, the target mRNA level is indicated as amass or percentage of target mRNA per total nucleic acids within thesample. In some embodiments, the target mRNA level is indicated relativeto another mRNA level, such as an mRNA level of a housekeeping gene,within the sample. In some embodiments, the target mRNA measurement isobtained by an assay such as a PCR assay. In some embodiments, the PCRcomprises qPCR. In some embodiments, the PCR comprises reversetranscription of the target mRNA.

In some embodiments, the composition reduces the target mRNA measurementrelative to the baseline target mRNA measurement. In some embodiments,the target mRNA measurement is obtained in a second sample obtained fromthe subject after administering the composition to the subject. In someembodiments, the composition reduces target mRNA levels relative to thebaseline target mRNA levels. In some embodiments, the reduced targetmRNA levels are measured in a second sample obtained from the subjectafter administering the composition to the subject. In some embodiments,the second sample is a second liver sample. In some embodiments, thesecond sample is second hepatocyte sample.

In some embodiments, the target mRNA measurement is reduced by about2.5% or more, about 5% or more, or about 7.5% or more, relative to thebaseline target mRNA measurement. In some embodiments, the target mRNAmeasurement is decreased by about 10% or more, relative to the baselinetarget mRNA measurement. In some embodiments, the target mRNAmeasurement is decreased by about 20% or more, about 30% or more, about40% or more, about 50% or more, about 60% or more, about 70% or more,about 80% or more, about 90% or more, or about 100% relative to thebaseline target mRNA measurement. In some embodiments, the target mRNAmeasurement is decreased by no more than about 2.5%, no more than about5%, or no more than about 7.5%, relative to the baseline target mRNAmeasurement. In some embodiments, the target mRNA measurement isdecreased by no more than about 10%, relative to the baseline targetmRNA measurement. In some embodiments, the target mRNA measurement isdecreased by no more than about 20%, no more than about 30%, no morethan about 40%, no more than about 50%, no more than about 60%, no morethan about 70%, no more than about 80%, no more than about 90%, or about100% relative to the baseline target mRNA measurement. In someembodiments, the target mRNA measurement is decreased by 2.5%, 5%, 7.5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by a range definedby any of the two aforementioned percentages. The target mRNAmeasurement may be decreased for an extended period of time. In someembodiments, the target mRNA measurement is decreased for 7 days, 14days, 28 days, 42 days, 56 days, 70 days, 77 days, 84 days, 91 days, 98days, 105 days, or a range therebetween. In some embodiments, the targetprotein measurement is decreased for about 7 days, about 14 days, about28 days, about 42 days, about 56 days, about 70 days, about 77 days,about 84 days, about 91 days, about 98 days, about 105 days, or a rangetherebetween. Some embodiments include decreasing an RNA measurementother than an mRNA measurement.

III. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

Throughout this application, various embodiments may be presented in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosure. Accordingly,the description of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a sample” includes a plurality ofsamples, including mixtures thereof.

Some examples relate to a sequence. To any extent that the sequencelisting contradicts the disclosure in the specification, thespecification takes precedent.

The terms “determining,” “measuring,” “evaluating,” “assessing,”“assaying,” and “analyzing” are often used interchangeably herein torefer to forms of measurement. The terms include determining if anelement is present or not (for example, detection). These terms caninclude quantitative, qualitative or quantitative and qualitativedeterminations. Assessing can be relative or absolute. “Detecting thepresence of” can include determining the amount of something present inaddition to determining whether it is present or absent depending on thecontext.

The terms “subject,” and “patient” may be used interchangeably herein. A“subject” can be a biological entity containing expressed geneticmaterials. The biological entity can be a plant, animal, ormicroorganism, including, for example, bacteria, viruses, fungi, andprotozoa. The subject can be a mammal. The mammal can be a human. Thesubject may be diagnosed or suspected of being at high risk for adisease. In some cases, the subject is not necessarily diagnosed orsuspected of being at high risk for the disease.

As used herein, the term “about” a number refers to that number plus orminus 10% of that number. The term “about” a range refers to that rangeminus 10% of its lowest value and plus 10% of its greatest value.

As used herein, the terms “treatment” or “treating” are used inreference to a pharmaceutical or other intervention regimen forobtaining beneficial or desired results in the recipient. Beneficial ordesired results include but are not limited to a therapeutic benefitand/or a prophylactic benefit. A therapeutic benefit may refer toeradication or amelioration of symptoms or of an underlying disorderbeing treated. Also, a therapeutic benefit can be achieved with theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the subject, notwithstanding that the subject may still beafflicted with the underlying disorder. A prophylactic effect includesdelaying, preventing, or eliminating the appearance of a disease orcondition, delaying or eliminating the onset of symptoms of a disease orcondition, slowing, halting, or reversing the progression of a diseaseor condition, or any combination thereof. For prophylactic benefit, asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease mayundergo treatment, even though a diagnosis of this disease may not havebeen made.

“Treatment” or “treating” may include an approach for obtainingbeneficial or desired results with respect to a disease, disorder, ormedical condition including but not limited to a therapeutic benefitand/or a prophylactic benefit. A therapeutic benefit can include, forexample, the eradication or amelioration of the underlying disorderbeing treated. Also, a therapeutic benefit can include, for example, theeradication or amelioration of one or more of the physiological symptomsassociated with the underlying disorder such that an improvement isobserved in the subject, notwithstanding that the subject may still beafflicted with the underlying disorder. In certain embodiments, forprophylactic benefit, the compositions are administered to a subject atrisk of developing a particular disease, or to a subject reporting oneor more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. Treatment viaadministration of a compound described herein does not necessarilyrequire the involvement of a medical professional.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

The term “C_(x-y)” or “C_(x)-C_(y)” when used in conjunction with achemical moiety, such as alkyl, alkenyl, or alkynyl is meant to includegroups that contain from x to y carbons in the chain. For example, theterm “C₁₋₆ alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from 1 to 6 carbons. The term “C_(x-y)” or“C_(x)-C_(y)” is not meant to limit the number of carbon atoms which maybe attached to the chemical moiety when the chemical moiety issubstituted with a second chemical moiety. For example, the term “C₁₋₆alkyl” or “C₁ to C₆ alkyl” refers to saturated, substituted orunsubstituted, hydrocarbon groups, including straight-chain alkyl groups(e.g., linear alkyl groups) and branched alkyl groups that contain 1, 2,3, 4, 5, or 6 carbon atoms, plus however many carbon atoms may bepresent in any substituents of the C₁₋₆ alkyl. For example, if a C₁₋₆alkyl is optionally substituted with a second chemical moiety comprisingtwo carbon atoms, then it will be understood that the C₁₋₆ alkyl caninclude between 1 and 8 carbon atoms.

The terms “C_(x-y) alkenyl” and “C_(x-y) alkynyl” refer to substitutedor unsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond, respectively.

“Amino” refers to the —NH₂ moiety.

“Cyano” refers to the —CN moiety.

“Nitro” refers to the —NO₂ moiety.

“Oxa” refers to the —O— moiety.

“Oxo” refers to the ═O moiety.

“Thioxo” refers to the ═S moiety.

“Imino” refers to the ═N—H moiety.

“Oximo” refers to the ═N—OH moiety.

“Hydrazino” refers to the ═N—NH₂ moiety.

“Alkyl” refers to a straight or branched hydrocarbon moiety consistingsolely of carbon and hydrogen atoms, fully saturated. In certainembodiments, “alkyl” comprises one to fifteen carbon atoms (e.g., C₁-C₁₅alkyl). In certain embodiments, an alkyl comprises one to thirteencarbon atoms (e.g., C₁-C₁₃ alkyl). In certain embodiments, an alkylcomprises one to eight carbon atoms (e.g., C₁-C₈ alkyl). In certainembodiments, an alkyl comprises one to six carbon atoms (e.g., C₁-C₆alkyl). In other embodiments, an alkyl comprises one to five carbonatoms (e.g., C₁-C₃ alkyl). In other embodiments, an alkyl comprises oneto four carbon atoms (e.g., C₁-C₄ alkyl). In other embodiments, an alkylcomprises one to three carbon atoms (e.g., C₁-C₃ alkyl). In otherembodiments, an alkyl comprises one to two carbon atoms (e.g., C₁-C₂alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g.,C₁ alkyl, e.g., methyl). In other embodiments, an alkyl comprises fiveto fifteen carbon atoms (e.g., C₅-C₁₅ alkyl). In other embodiments, analkyl comprises five to eight carbon atoms (e.g., C₅-C₈alkyl). In otherembodiments, an alkyl comprises two to five carbon atoms (e.g., C₂-C₈alkyl). In other embodiments, an alkyl comprises three to five carbonatoms (e.g., C₃-C₈ alkyl). In other embodiments, the alkyl group isselected from methyl, ethyl, 1-propyl (n-propyl), I-methylethyl(2-propyl, iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl),2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl(n-pentyl). The alkyl is attached to the rest of the molecule by asingle bond.

“Aminoalkyl” refers to a moiety boded through a nitrogen atom of theform —N(H)(alkyl) or N(alkyl)(alkyl), wherein when the moiety isN(alkyl)(alkyl), the two alkyl groups bonded to nitrogen can be the samealkyl groups or different alkyl groups.

“Alkoxy” refers to a moiety bonded through an oxygen atom of the formula—O-alkyl, where alkyl is an alkyl chain as defined above.

“Alkenyl” refers to a straight or branched hydrocarbon moiety consistingsolely of carbon and hydrogen atoms, containing at least onecarbon-carbon double bond. In certain embodiments, an alkenyl comprisestwo to twelve carbon atoms. In certain embodiments, an alkenyl comprisestwo to eight carbon atoms. In other embodiments, an alkenyl comprisestwo to four carbon atoms. The alkenyl is attached to the rest of themolecule by a single bond, for example, ethenyl (i.e., vinyl),prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl,and the like.

“Alkynyl” refers to a straight or branched hydrocarbon moiety consistingsolely of carbon and hydrogen atoms, containing at least onecarbon-carbon triple bond, having from two to twelve carbon atoms, andoptionally further comprising at least one carbon-carbon double bond. Incertain embodiments, an alkynyl comprises two to eight carbon atoms. Inother embodiments, an alkynyl comprises two to six carbon atoms. Inother embodiments, an alkynyl comprises two to four carbon atoms. Thealkynyl is attached to the rest of the molecule by a single bond, forexample, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

“Alkylene” or “alkylene chain” refers to a linear (e.g., straight), orbranched, divalent, hydrocarbon moiety. An “alkylene” or “alkylenechain” can link a portion of the molecule to a second moiety. An“alkylene” or “alkylene chain” consists solely of carbon and hydrogenatoms (substitution of an alkylene with one or more substituentscomprising atoms other than hydrogen, such as N, O, and S, may bespecified). An “alkylene” or “alkylene chain” can contain nounsaturation (notwithstanding the points of attachment of an alkylene tothe rest of the molecule). In certain embodiments, the “alkylene” or“alkylene chain” and comprises one to twelve carbon atoms, for example,methylene, ethylene, propylene, n-butylene, and the like. The alkylenechain can be attached to the portion of the molecule through a singlebond and to the second moiety through a single bond. The points ofattachment of an alkylene chain to the rest of the molecule and to thesecond moiety can be through one carbon in the alkylene chain or throughany two carbons within the alkylene. In certain embodiments, an alkylenecomprises one to eight carbon atoms (e.g., C₁-C₈ alkylene). In otherembodiments, an alkylene comprises one to five carbon atoms (e.g., C₁-C₃alkylene). In other embodiments, an alkylene comprises one to fourcarbon atoms (e.g., C₁-C₄ alkylene). In other embodiments, an alkylenecomprises one to three carbon atoms (e.g., C₁-C₃ alkylene). In otherembodiments, an alkylene comprises one to two carbon atoms (e.g., C₁-C₂alkylene). In other embodiments, an alkylene comprises one carbon atom(e.g., C₁ alkylene). In other embodiments, an alkylene comprises five toeight carbon atoms (e.g., C₅-C₈ alkylene). In other embodiments, analkylene comprises two to five carbon atoms (e.g., C₂-C₅ alkylene). Inother embodiments, an alkylene comprises three to five carbon atoms(e.g., C₃-C₅ alkylene).

“Alkenylene” or “alkenylene chain” refers to a linear (e.g., straight),or branched, divalent, hydrocarbon moiety. An “alkenylene” or“alkenylene chain” can link a portion of the molecule to a secondmoiety. An “alkenylene” or “alkenylene chain” consists solely of carbonand hydrogen atoms (substitution of an alkenylene with one or moresubstituents comprising atoms other than hydrogen, such as N, O, and S,may be specified). An “alkenylene” or “alkenylene chain” comprises atleast one carbon-carbon double bond. In certain embodiments, an“alkenylene” or “alkenylene chain” comprises from two to twelve carbonatoms. The alkenylene chain can be attached to the portion of themolecule through a single bond and to the second moiety through a singlebond. The points of attachment of an alkenylene chain to the rest of themolecule and to the second moiety can be through one carbon in thealkenylene chain or through any two carbons within the alkenylene chain.In certain embodiments, an alkenylene comprises two to eight carbonatoms (e.g., C₂-C₈ alkenylene). In other embodiments, an alkenylenecomprises two to five carbon atoms (e.g., C₂-C₈ alkenylene). In otherembodiments, an alkenylene comprises two to four carbon atoms (e.g.,C₂-C₄ alkenylene). In other embodiments, an alkenylene comprises two tothree carbon atoms (e.g., C₂-C₃ alkenylene). In other embodiments, analkenylene comprises five to eight carbon atoms (e.g., C₅-C₈alkenylene).In other embodiments, an alkenylene comprises two to five carbon atoms(e.g., C₂-C₅ alkenylene). In other embodiments, an alkenylene comprisesthree to five carbon atoms (e.g., C₃-C₅ alkenylene).

“Alkynylene” or “alkynylene chain” refers to a linear (e.g., straight),or branched, divalent, hydrocarbon moiety. An “alkynylene” or“alkynylene chain” can link a portion of the molecule to a secondmoiety. An “alkynylene” or “alkynylene chain” consists solely of carbonand hydrogen (substitution of an alkynylene with one or moresubstituents comprising atoms other than hydrogen, such as N, O, and S,may be specified). An “alkynylene” or “alkynylene chain” comprises atleast one carbon-carbon triple bond. In certain embodiments, an“alkynylene” or “alkynylene chain” comprises from two to twelve carbonatoms. An alkynylene chain can be attached to the portion of themolecule through a single bond and to the second moiety through a singlebond. The points of attachment of an alkynylene chain to the rest of themolecule and to the second moiety can be through one carbon in thealkynylene chain or through any two carbons within the alkynylene chain.In certain embodiments, an alkynylene comprises two to eight carbonatoms (e.g., C₂-C₈ alkynylene). In other embodiments, an alkynylenecomprises two to five carbon atoms (e.g., C₂-C₅ alkynylene). In otherembodiments, an alkynylene comprises two to four carbon atoms (e.g.,C₂-C₄ alkynylene). In other embodiments, an alkynylene comprises two tothree carbon atoms (e.g., C₂-C₃ alkynylene). In other embodiments, analkynylene comprises two carbon atom (e.g., C₂ alkylene). In otherembodiments, an alkynylene comprises five to eight carbon atoms (e.g.,C₅-C₈ alkynylene). In other embodiments, an alkynylene comprises threeto five carbon atoms (e.g., C₃-C₃ alkynylene).

The term “carbocycle” as used herein refers to a saturated, unsaturatedor aromatic ring in which each atom of the ring is carbon. Carbocycleincludes 3- to 10-membered monocyclic rings, 5- to 12-membered bicyclicrings, 5- to 12-membered spiro bicycles, and 5- to 12-membered bridgedrings. Each ring of a bicyclic carbocycle may be selected fromsaturated, unsaturated, and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Abicyclic carbocycle includes any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits. A bicyclic carbocyclefurther includes spiro bicyclic rings such as spiropentane. A bicycliccarbocycle includes any combination of ring sizes such as 3-3 spiro ringsystems, 4-4 spiro ring systems, 4-5 fused ring systems, 5-5 fused ringsystems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ringsystems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fusedring systems. Exemplary carbocycles include cyclopentyl, cyclohexyl,cyclohexenyl, adamantyl, phenyl, indanyl, naphthyl, andbicyclo[1.1.1]pentanyl. “Carbocycle” may include “aryl” and“cycloalkyl.”

The term “aryl” refers to an aromatic monocyclic or aromatic multicyclichydrocarbon ring system. The aromatic monocyclic or aromatic multicyclichydrocarbon ring system contains only hydrogen and carbon and from fiveto eighteen carbon atoms, where at least one of the rings in the ringsystem is aromatic, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. The ring systemfrom which aryl groups are derived include, but are not limited to,groups such as benzene, fluorene, indane, indene, tetralin andnaphthalene. In some embodiments, the aryl substituent is positively ornegatively charged. In some embodiments, the aryl substituent isneutral. In some embodiments, the aryl substituent is zwitterionic;alternatively, or in addition, in some embodiments, the aryl substituentis not charged. In some embodiments, the aryl substituent bears nocharges. In some embodiments, the aryl substituent bears no net charge.In some embodiments, the aryl substituent bears no net charge and is notzwitterionic.

The term “cycloalkyl” refers to a saturated ring in which each atom ofthe ring is carbon. Cycloalkyl may include monocyclic and polycyclicrings such as 3- to 10-membered monocyclic rings, 5- to 12-memberedbicyclic rings, 5- to 12-membered spiro bicycles, and 5- to 12-memberedbridged rings. In certain embodiments, a cycloalkyl comprises three toten carbon atoms. In other embodiments, a cycloalkyl comprises five toseven carbon atoms. The cycloalkyl may be attached to the rest of themolecule by a single bond. Examples of monocyclic cycloalkyls include,e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Polycyclic cycloalkyl radicals include, for example,adamantyl, spiropentane, norbornyl (i.e., bicyclo[2.2.1]heptanyl),decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, bicyclo[1.1.1]pentanyl,and the like.

The term “cycloalkenyl” refers to a saturated ring in which each atom ofthe ring is carbon and there is at least one double bond between tworing carbons. Cycloalkenyl may include monocyclic and polycyclic ringssuch as 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclicrings, and 5- to 12-membered bridged rings. In other embodiments, acycloalkenyl comprises five to seven carbon atoms. The cycloalkenyl maybe attached to the rest of the molecule by a single bond. Examples ofmonocyclic cycloalkenyls include, e.g., cyclopentenyl, cyclohexenyl,cycloheptenyl, and cyclooctenyl.

The term “halo” or, alternatively, “halogen” or “halide,” means fluoro,chloro, bromo or iodo. In some embodiments, halo is fluoro, chloro, orbromo.

The term “haloalkyl” refers to an alkyl radical, as defined above, thatis substituted by one or more halo radicals, for example,trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl,1-chloromethyl-2-fluoroethyl, and the like. In some embodiments, thealkyl part of the haloalkyl radical is optionally further substituted asdescribed herein.

The term “heterocycle” as used herein refers to a saturated, unsaturatedor aromatic ring comprising one or more heteroatoms. Exemplaryheteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3-to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, 5- to12-membered spiro bicycles, and 5- to 12-membered bridged rings. Abicyclic heterocycle includes any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits. In an exemplaryembodiment, an aromatic ring, e.g., pyridyl, may be fused to a saturatedor unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine,piperidine or cyclohexene. A bicyclic heterocycle includes anycombination of ring sizes such as 4-5 fused ring systems, 5-5 fused ringsystems, 5-6 fused ring systems, 6-6 fused ring systems, 5-7 fused ringsystems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fusedring systems. A bicyclic heterocycle further includes spiro bicyclicrings, e.g., 5 to 12-membered spiro bicycles, such as2-oxa-6-azaspiro[3.3]heptane. “Heterocycle” may include “heteroaryl” and“heterocycloalkyl”.

The term “heteroaryl” refers to a radical derived from a 5 to 18membered aromatic ring radical that comprises two to seventeen carbonatoms and from one to six heteroatoms selected from nitrogen, oxygen andsulfur. As used herein, the heteroaryl radical is a monocyclic,bicyclic, tricyclic or tetracyclic ring system, wherein at least one ofthe rings in the ring system is aromatic, i.e., it contains a cyclic,delocalized (4n+2) π-electron system in accordance with the Hückeltheory. Heteroaryl includes fused or bridged ring systems. Theheteroatom(s) in the heteroaryl radical is optionally oxidized. One ormore nitrogen atoms, if present, are optionally quaternized. Theheteroaryl is attached to the rest of the molecule through any atom ofthe ring(s). Examples of heteroaryls include, but are not limited to,azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl,benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl,pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl,pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl,quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e.thienyl).

One or more nitrogen atoms, if present, may be optionally quaternized.In some embodiments, the heterocycle substituent is positively ornegatively charged. In some embodiments, the heterocycle substituent isneutral. In some embodiments, the heterocycle substituent iszwitterionic; alternatively, or in addition, in some embodiments, theheterocycle substituent is not charged. In some embodiments, theheterocycle substituent bears no charges. In some embodiments, theheterocycle substituent bears no net charge. In some embodiments, theheterocycle substituent bears no net charge and is not zwitterionic.

The term “heterocycloalkyl” refers to a saturated ring with carbon atomsand at least one heteroatom. Exemplary heteroatoms include N, O, Si, P,B, and S atoms. Heterocycloalkyl may include monocyclic and polycyclicrings such as 3- to 10-membered monocyclic rings, 6- to 12-memberedbicyclic rings, 5- to 12-membered spiro bicycles, and 5- to 12-memberedbridged rings. The heteroatoms in the heterocycloalkyl radical areoptionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heterocycloalkyl is attached to the rest ofthe molecule through any atom of the heterocycloalkyl, valencepermitting, such as any carbon or nitrogen atoms of theheterocycloalkyl. Examples of heterocycloalkyl radicals include, but arenot limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,2-oxa-6-azaspiro[3.3]heptane, and 1,1-dioxo-thiomorpholinyl. In someembodiments, a heterocycloalkyl comprises one heteroatom. In someembodiments, a heterocycloalkyl comprises one heteroatom selected fromN, O, and S. In some embodiments, a heterocycloalkyl comprises multipleheteroatoms. In some embodiments, a heterocycloalkyl comprises multipleheteroatoms selected from N, O, and S.

The term “heterocycloalkenyl” refers to an unsaturated ring with carbonatoms and at least one heteroatom and there is at least one double bondbetween two ring carbons. Heterocycloalkenyl does not include heteroarylrings. Exemplary heteroatoms include N, O, Si, P. B, and S atoms.Heterocycloalkenyl may include monocyclic and polycyclic rings such as3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings,and 5- to 12-membered bridged rings. In other embodiments, aheterocycloalkenyl comprises five to seven ring atoms. Theheterocycloalkenyl may be attached to the rest of the molecule by asingle bond. Examples of monocyclic cycloalkenyls include, e.g.,pyrroline (dihydropyrrole), pyrazoline (dihydropyrazole), imidazoline(dihydroimidazole), triazoline (dihydrotriazole), dihydrofuran,dihydrothiophene, oxazoline (dihydrooxazole), isoxazoline(dihydroisoxazole), thiazoline (dihydrothiazole), isothiazoline(dihydroisothiazole), oxadiazoline (dihydrooxadiazole), thiadiazoline(dihydrothiadiazole), dihydropyridine, tetrahydropyridine,dihydropyridazine, tetrahydropyridazine, dihydropyrimidine,tetrahydropyrimidine, dihydropyrazine, tetrahydropyrazine, pyran,dihydropyran, thiopyran, dihydrothiopyran, dioxine, dihydrodioxine,oxazine, dihydrooxazine, thiazine, and dihydrothiazine.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons or substitutable heteroatoms, e.g., anNH or NH₂ of a compound. It will be understood that “substitution” or“substituted with” includes the implicit proviso that such substitutionis in accordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,i.e., a compound which does not spontaneously undergo transformationsuch as by rearrangement, cyclization, elimination, etc. In certainembodiments, substituted refers to moieties having substituentsreplacing two hydrogen atoms on the same carbon atom, such assubstituting the two hydrogen atoms on a single carbon with an oxo,imino or thioxo group. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds.

In some embodiments, substituents may include any substituents describedherein, for example: halogen, hydroxy, oxo (═O), thioxo (═S), cyano(—CN), nitro (—NO₂), imino (═N—H), oximo (═N—OH), hydrazino (═N—NH₂),—R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR,—R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(b)—C(O)N(R^(a))₂,—R^(b)—N(R)C(O)OR^(a), —R^(b)—N(R)C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2), and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); and alkyl, alkenyl,alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl,and heteroarylalkyl, any of which may be optionally substituted byalkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl,oxo (═O), thioxo (═S), cyano (—CN), nitro (—NO₂), imino (═N—H), oximo(═N—OH), hydrazine (═N—NH₂), —R^(b)—OR^(a), —R^(b)—OC(O)—R^(a),—R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂,—R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂,—R^(b)—O—R^(c)—C(O)N(R^(a))₂, —R^(b)—N(R^(a))C(O)OR^(a),—R^(b)—N(R^(a))C(O)R^(a), —R^(b)—N(R^(a))S(O)R^(a) (where t is 1 or 2),—R^(b)—S(O)_(t)R (where t is 1 or 2), —R^(b)—S(O)OR^(a) (where t is 1 or2) and —R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); wherein each R^(a)is independently selected from hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl,heteroaryl, or heteroarylalkyl, wherein each R, valence permitting, maybe optionally substituted with alkyl, alkenyl, alkynyl, halogen,haloalkyl, haloalkenyl, haloalkynyl, oxo (═O), thioxo (═S), cyano (—CN),nitro (—NO₂), imino (═N—H), oximo (═N—OH), hydrazine (═N—NH₂),—R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a),—R^(b)—OC(O)—N(R)₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R—C(O)N(R^(b))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(b))C(O)R^(a),—R^(b)—N(R^(a))S(O)R (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where tis 1 or 2), —R^(b)—S(O)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2); and wherein each R^(b) isindependently selected from a direct bond or a straight or branchedalkylene, alkenylene, or alkynylene chain, and each R^(c) is a straightor branched alkylene, alkenylene or alkynylene chain.

Double bonds to oxygen atoms, such as oxo groups, are represented hereinas both “═O” and “(O)”. Double bonds to nitrogen atoms are representedas both “═NR” and “(NR)”. Double bonds to sulfur atoms are representedas both “═S” and “(S)”.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate: (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

The term “salt” or “pharmaceutically acceptable salt” refers to saltsderived from a variety of organic and inorganic counter ions well knownin the art. Pharmaceutically acceptable acid addition salts can beformed with inorganic acids and organic acids. Inorganic acids fromwhich salts can be derived include, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike. Organic acids from which salts can be derived include, forexample, acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid,salicylic acid, and the like. Pharmaceutically acceptable base additionsalts can be formed with inorganic and organic bases. Inorganic basesfrom which salts can be derived include, for example, sodium, potassium,lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like. Organic bases from which salts can be derivedinclude, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. In some embodiments, thepharmaceutically acceptable base addition salt is chosen from ammonium,potassium, sodium, calcium, and magnesium salts.

VI. EXAMPLES Example 1: Identification of Variants in a TargetOligonucleotide Associated with Increased or Decreased Risk of aDisorder

Approximately 30,000,000 imputed variants are to be analyzed in ˜375,000individuals from a Biobank cohort for associations with liver disorderssuch as non-alcoholic steatohepatitis.

Protective or maladaptive associations are observed between specificallelic variants of various target genes and liver diseases. Theassociations will suggest that in some cases therapeutic inhibition ormodulation of a target protein encoded by any of the target genes may bean effective genetically-informed method of treatment for any of theliver disorders.

Example 2: Bioinformatic Selection of Sequences in Order to IdentifyTherapeutic siRNAs to Downmodulate Expression of a Target mRNA

Screening sets are to be defined based on bioinformatic analysis.Therapeutic siRNAs are designed to bind human target mRNAs, and thetarget mRNA sequence of at least one toxicology-relevant species such asa non-human primate (NHP) species (e.g. a rhesus or cynomolgus monkey).Drivers for the design of the screening set are predicted specificity ofthe siRNAs against the transcriptome of the relevant species as well ascross-reactivity between species. Predicted specificity in human, rhesusmonkey, cynomolgus monkey, mouse and rat are determined for sense (S)and antisense (AS) strands. These are assigned a “specificity score”which considers the likelihood of unintended downregulation of any othertranscript by full or partial complementarity of an siRNA strand (up to4 mismatches within positions 2-18) as well as the number and positionsof mismatches. Thus, off-target(s) for antisense and sense strands ofeach siRNA are identified. In addition, the number of potentialoff-targets are used as an additional specificity factor in thespecificity score. As identified, siRNAs with high specificity and a lownumber of predicted off-targets provide a benefit of increased targetingspecificity.

In addition to selecting siRNA sequences with high sequence specificityto the target mRNA, siRNA sequences within a seed region are analyzedfor similarity to seed regions of known miRNAs, siRNAs can function in amiRNA like manner via base-pairing with complementary sequences withinthe Y-UTR of mRNA molecules. The complementarity typically encompassesthe 5′-bases at positions 2-7 of the miRNA (seed region). To circumventsiRNAs to act via functional miRNA binding sites, siRNA strandscontaining natural miRNA seed regions are avoided. Seed regionsidentified in miRNAs from human, mouse, rat, rhesus monkey, dog, rabbitand pig are referred to as “conserved”. Combining the “specificityscore” with miRNA seed analysis yields a “specificity category”. This isdivided into categories 1-4, with 1 having the highest specificity and 4having the lowest specificity. Each strand of the siRNA is assigned to aspecificity category.

Species cross-reactivity are assessed for human, cynomolgus monkey,rhesus monkey, mouse and rat. The analysis is based on a canonical siRNAdesign using 19 bases and 17 bases (without considering positions 1 and19) for cross-reactivity. Full match as well as single mismatch analysesare included.

Analysis of the human Single Nucleotide Polymorphism (SNP) database(NCBI-DB-SNP) to identify siRNAs targeting regions with known SNPs arealso carried out to identify siRNAs that may be non-functional inindividuals containing the SNP. Information regarding the positions ofSNPs within the target sequence as well as minor allele frequency (MAF)in case data are obtained in this analysis.

The above methods can be used to identify therapeutic siRNAs todownmodulate expression of a target mRNA. Bioinformatic methods may alsobe used to identify ASOs that bind and downmodulate expression of atarget mRNA.

Example 3: Chemically Modified siRNAs

siRNAs that bind a target mRNA can be synthesized with chemicalmodifications with the sense strand having a modification such asmodification pattern 1S, and the antisense strand having a modificationsuch as modification pattern 1AS. In addition, adenosine can be placedat position 19 in the sense strand and uridine at position 1 in theantisense strand.

The siRNAs that bind a target mRNA can also be synthesized with chemicalmodifications with the sense strand having modification pattern 2S andthe antisense strand having modification pattern 3AS. In addition,adenosine can be placed at position 19 in the sense strand and uridineat position 1 in the antisense strand.

The siRNAs that bind a target mRNA can also be synthesized with chemicalmodifications with the sense strand having modification pattern 2S andthe antisense strand having modification pattern 9AS. In addition,adenosine can be placed at position 19 in the sense strand and uridineat position 1 in the antisense strand.

The siRNAs targeting that bind a target mRNA can also be synthesizedwith chemical modifications with the sense strand having modificationpattern 3S and the antisense strand having modification pattern 3AS. Inaddition, adenosine can be placed at position 19 in the sense strand anduridine at position 1 in the antisense strand.

The siRNAs targeting that bind a target mRNA can also be synthesizedwith chemical modifications with the sense strand having any of thefollowing: all purines comprising 2′ fluoro modified purines, and allpyrimidines comprising a mixture of 2′ fluoro and 2′-O-methyl modifiedpyrimidines; all purines comprising 2′-O-methyl modified purines, andall pyrimidines comprising a mixture of 2′ fluoro and 2′-O-methylmodified pyrimidines; all purines comprising 2′ fluoro modified purines,and all pyrimidines comprising 2′-O-methyl modified pyrimidines; allpyrimidines comprising 2′ fluoro modified pyrimidines, and all purinescomprising a mixture of 2′ fluoro and 2′-O-methyl modified purines; allpyrimidines comprising 2′-O-methyl modified pyrimidines, and all purinescomprising a mixture of 2′ fluoro and 2′-O-methyl modified purines; orall pyrimidines comprising 2′ fluoro modified pyrimidines, and allpurines comprising 2′-O-methyl modified purines; and further with theantisense strand having any of the following: all purines comprising 2′fluoro modified purines, and all pyrimidines comprising a mixture of 2′fluoro and 2′-O-methyl modified pyrimidines; all purines comprising2′-O-methyl modified purines, and all pyrimidines comprising a mixtureof 2′ fluoro and 2′-O-methyl modified pyrimidines; all purinescomprising 2′-O-methyl modified purines, and all pyrimidines comprising2′ fluoro modified pyrimidines; all pyrimidines comprising 2′ fluoromodified pyrimidines, and all purines comprising a mixture of 2′ fluoroand 2′-O-methyl modified purines; all pyrimidines comprising 2′-O-methylmodified pyrimidines, and all purines comprising a mixture of 2′ fluoroand 2′-O-methyl modified purines; or all pyrimidines comprising2′-O-methyl modified pyrimidines, and all purines comprising 2′ fluoromodified purines.

Example 4: Screening siRNAs for Activity in Cells in Culture

The chemically modified siRNAs derived from sequences in the previousExamples will be assayed for target mRNA knockdown activity in cells inculture. A cell line that expresses the target mRNA is to be seeded in96-well tissue culture plates at a cell density of 10,000 cells per wellin DMEM supplemented with 10% fetal bovine serum and incubated overnightin a water-jacketed, humidified incubator at 37° C. in an atmospherecomposed of air plus 5% carbon dioxide. The siRNAs are individuallytransfected into cells in duplicate wells at 10 nM final concentrationusing 0.3 μL Lipofectamine RNAiMax (Fisher) per well. Silencer SelectNegative Control #1 (ThermoFisher, Catalog #4390843) and a positivecontrol siRNA are transfected at 10 nM final concentration as controls.After incubation for 48 hours at 37° C., total RNA is harvested fromeach well and cDNA prepared using TaqMan® Fast Advanced Cells- to-CT-mKit (ThermoFisher, Catalog #A35374) according to the manufacturer'sinstructions. The level of target mRNA in each well will be measured intriplicate by real-time qPCR on an Applied Biosystems 7500 FastReal-Time PCR machine using TaqMan Gene Expression Assay for the humantarget mRNA. The level of PPIA mRNA will be measured using TaqMan GeneExpression Assay (ThermoFisher) and used to determine relative targetmRNA levels in each well using the delta-delta Ct method. Data will benormalized to relative target mRNA levels in untreated cells.

The siRNAs showing the greatest degree of knockdown of target mRNA at 10nM will be tested in a second screen for activity at 1 nM concentrationusing the transfection procedures as described above. Similarexperiments may be performed using ASOs. Thus, siRNAs and ASOs may beidentified that most effectively downmodulate expression of the targetmRNA.

Example 5: GalNAc Ligands for Hepatocyte Targeting of Oligonucleotides

Without limiting the disclosure to these individual methods, there areat least two general methods for attachment of multivalentN-acetylgalactosamine (GalNAc) ligands to oligonucleotides; solid orsolution-phase conjugations. GalNAc ligands may be attached to solidphase resin for 3′ conjugation or at the 5′ terminus using GalNAcphosphoramidite reagents. GalNAc phosphoramidites may be coupled onsolid phase as for other nucleosides in the oligonucleotide sequence atany position in the sequence. A non-limiting example of aphosphoramidite reagent for GalNAc conjugation to a 5′ endoligonucleotide is shown in Table 1.

TABLE 1 GalNAc Conjugation Reagent Type of conjugation Structure Solidphase 5′ attachment phosphoramidite

Example 6: Synthesis of GalNAc Ligands Scheme for the Preparation ofNAcegal-Linker-TMSOTf

General Procedure for Preparation of Compound 2A

To a solution of Compound 1A (500 g, 4.76 mol, 476 mL) in 2-Methly-THF(2.00 L) was added CbzCl (406 g, 2.38 mol, 338 mL) in 2-Methyl-THF (750mL) dropwise at 0° C. The mixture was stirred at 25° C. for 2 hrs underN₂ atmosphere. TLC (DCM:MeOH=20:1, PMA) indicated CbzCl was consumedcompletely and one new spot (R_(f)=0.43) formed. The reaction mixturewas added HCl/EtOAc (1 N, 180 mL) and stirred for 30 mins, white solidwas removed by filtration through celite, the filtrate was concentratedunder vacuum to give Compound 2A (540 g, 2.26 mol, 47.5% yield) as apale yellow oil and used into the next step without furtherpurification. ¹H NMR: δ 7.28-7.41 (m, 5H), 5.55 (br s, 1H), 5.01-5.22(m, 2H), 3.63-3.80 (m, 2H), 3.46-3.59 (m, 4H), 3.29-3.44 (m, 2H),2.83-3.02 (m, 1H).

General Procedure for Preparation of Compound 4A

To a solution of Compound 3A (1.00 kg, 4.64 mol, HCl) in pyridine (5.00L) was added acetyl acetate (4.73 kg, 46.4 mol, 4.34 L) dropwise at 0°C. under N₂ atmosphere. The mixture was stirred at 25° C. for 16 hrsunder N₂ atmosphere. TLC (DCM:MeOH=20:1, PMA) indicated Compound 3A wasconsumed completely and two new spots (R_(f)=0.35) formed. The reactionmixture was added to cold water (30.0 L) and stirred at 0° C. for 0.5hr, white solid formed, filtered and dried to give Compound 4A (1.55 kg,3.98 mol, 85.8% yield) as a white solid and used in the next stepwithout further purification. ¹H NMR: δ 7.90 (d, J=9.29 Hz, 1H), 5.64(d, J=8.78 Hz, 1H), 5.26 (d, J=3.01 Hz, 1H), 5.06 (dd, J=11.29, 3.26 Hz,1H), 4.22 (t, J=6.15 Hz, 1H), 3.95-4.16 (m, 3H), 2.12 (s, 3H), 2.03 (s,3H), 1.99 (s, 3H), 1.90 (s, 3H), 1.78 (s, 3H).

General Procedure for Preparation of Compound 5A

To a solution of Compound 4A (300 g, 771 mmol) in DCE (1.50 L) was addedTMSOTf (257 g, 1.16 mol, 209 mL) and stirred for 2 hrs at 60° C., andthen stirred for 1 hr at 25° C. Compound 2A (203 g, 848 mmol) wasdissolved in DCE (1.50 L) and added 4 Å powder molecular sieves (150 g)stirring for 30 mins under N₂ atmosphere. Then the solution of Compound4A in DCE was added dropwise to the mixture at 0° C. The mixture wasstirred at 25° C. for 16 hrs under N₂ atmosphere. TLC (DCM:MeOH=25:1,PMA) indicated Compound 4A was consumed completely and new spot(R_(f)=0.24) formed. The reaction mixture was filtered and washed withsat. NaHCO₃ (2.00 L), water (2.00 L) and sat. brine (2.00 L). Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to give a residue. The residue was trituratedwith 2-Me-THE/heptane (5/3, v/v, 1.80 L) for 2 hrs, filtered and driedto give Compound 5A (225 g, 389 mmol, 50.3% yield, 98.4% purity) as awhite solid. ¹H NMR: δ 7.81 (d, J=9.29 Hz, 1H), 7.20-7.42 (m, 6H), 5.21(d, J=3.26 Hz, 1H), 4.92-5.05 (m, 3H), 4.55 (d, J=8.28 Hz, 1H),3.98-4.07 (m, 3H), 3.82-3.93 (m, 1H), 3.71-3.81 (m, 1H), 3.55-3.62 (m,1H), 3.43-3.53 (m, 2H), 3.37-3.43 (m, 2H), 3.14 (q, J=5.77 Hz, 2H), 2.10(s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H).

General Procedure for Preparation of NAcegal-Linker-Tosylate Salt

To a solution of Compound 5A (200 g, 352 mmol) in THF (1.0 L) was addeddry Pd/C (15.0 g, 10% purity) and TsOH (60.6 g, 352 mmol) under N₂atmosphere. The suspension was degassed under vacuum and purged with H₂several times. The mixture was stirred at 25° C. for 3 hrs under H₂ (45psi) atmosphere. TLC (DCM:MeOH=10:1, PMA) indicated Compound 5A wasconsumed completely and one new spot (Rt=0.04) was formed. The reactionmixture was filtered and concentrated (40° C.) under reduced pressure togive a residue. Diluted with anhydrous DCM (500 mL, dried overnight with4 Å molecular sieves (dried at 300° C. for 12 hrs)) and concentrate togive a residue and run Karl Fisher (KF) to check for water content. Thiswas repeated 3 times with anhydrous DCM (500 mL) dilutions andconcentration to give NAcegal-Linker-TMSOTf (205 g, 95.8% yield, TsOHsalt) as a foamy white solid. ¹H NMR: δ 7.91 (d, J=9.03 Hz, 1H),7.53-7.86 (m, 2H), 7.49 (d, J=8.03 Hz, 2H), 7.13 (d, J=8.03 Hz, 2H),5.22 (d, J=3.26 Hz, 1H), 4.98 (dd, J=11.29, 3.26 Hz, 1H), 4.57 (d,J=8.53 Hz, 1H), 3.99-4.05 (m, 3H), 3.87-3.94 (m, 1H), 3.79-3.85 (m, 1H),3.51-3.62 (m, 5H), 2.96 (br t, J=5.14 Hz, 2H), 2.29 (s, 3H), 2.10 (s,3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.78 (s, 3H).

General Procedure for Preparation of Compound 5B

To a solution of Compound 4B (400 g, 1.67 mol, 1.00 eq) and NaOH (10 M,16.7 mL, 0.10 eq) in THF (2.00 L) was added Compound 4B_2 (1.07 kg, 8.36mol, 1.20 L, 5.00 eq), the mixture was stirred at 30° C. for 2 hrs. LCMSshowed the desired MS was given. Five batches of solution were combinedto one batch, then the mixture was diluted with water (6.00 L),extracted with ethyl acetate (3.00 L*3), the combined organic layer waswashed with brine (3.00 L), dried over Na₂SO₄, filtered and concentratedunder vacuum. The crude was purified by column chromatography (SiO₂,petroleum ether:ethyl acetate=100:1-10:1, R=0.5) to give Compound 5B(2.36 kg, 6.43 mol, 76.9% yield) as light yellow oil. HNMR: δ 7.31-7.36(m, 5H), 5.38 (s, 1H), 5.11-5.16 (m, 2H), 3.75 (t, J=6.4 Hz), 3.54-3.62(m, 6H), 3.39 (d, J=5.2 Hz), 2.61 (t, J=6.0 Hz).

General Procedure for Preparation of3-oxo-1-phenyl-2,7,10-trioxa-4-azatridecan-13-oic acid (Compound 2Bbelow)

To a solution of Compound 5B (741 g, 2.02 mol, 1.00 eq) in DCM (2.80 L)was added TFA (1.43 kg, 12.5 mol, 928 mL, 6.22 eq), the mixture wasstirred at 25° C. for 3 hrs. LCMS showed the desired MS was given. Themixture was diluted with DCM (5.00 L), washed with water (3.00 L*3),brine (2.00 L), the combined organic layer was dried over Na₂SO₄,filtered and concentrated under vacuum to give Compound 2B (1800 g,crude) as light yellow oil. HNMR: δ 9.46 (s, 5H), 7.27-7.34 (m, 5H),6.50-6.65 (m, 1H), 5.71 (s, 1H), 5.10-5.15 (m, 2H), 3.68-3.70 (m, 14H),3.58-3.61 (m, 6H), 3.39 (s, 2H), 2.55 (s, 6H), 2.44 (s, 2H).

General Procedure for Preparation of Compound 3B

To a solution of Compound 2B (375 g, 999 mmol, 83.0% purity, 1.00 eq) inDCM (1.80 L) was added HATU (570 g, 1.50 mol, 1.50 eq) and DIEA (258 g,2.00 mol, 348 mL, 2.00 eq) at 0° C., the mixture was stirred at 0° C.for 30 min, then Compound 1B (606 g, 1.20 mol, 1.20 eq) was added, themixture was stirred at 25° C. for 1 hr. LCMS showed desired MS wasgiven. The mixture was combined to one batch, then the mixture wasdiluted with DCM (5.00 L), washed with 1 N HCl aqueous solution (2.00L*2), then the organic layer was washed with saturated Na₂CO₃ aqueoussolution (2.00 L*2) and brine (2.00 L), the organic layer was dried overNa₂SO₄, filtered and concentrated under vacuum to give Compound 3B (3.88kg, crude) as yellow oil.

General Procedure for Preparation of TRIS-PEG2-CBZ

A solution of Compound 3B (775 g, 487 mmol, 50.3% purity, 1.00 eq) inHC/dioxane (4 M, 2.91 L, 23.8 eq) was stirred at 25° C. for 2 hrs. LCMSshowed the desired MS was given. The mixture was concentrated undervacuum to give a residue. Then the combined residue was diluted with DCM(5.00 L), adjusted to pH=8 with 2.5 M NaOH aqueous solution, andseparated. The aqueous phase was extracted with DCM (3.00 L) again, thenthe aqueous solution was adjusted to pH=3 with 1 N HCl aqueous solution,then extracted with DCM (5.00 L*2), the combined organic layer waswashed with brine (3.00 L), dried over Na₂SO₄, filtered and concentratedunder vacuum. The crude was purified by column chromatography (SiO₂,DCM:MeOH=0:1-12:1, 0.1% HOAc, R=0.4). The residue was diluted with DCM(5.00 L), adjusted to pH=8 with 2.5 M NaOH aqueous solution, separated,the aqueous solution was extracted with DCM (3.00 L) again, then theaqueous solution was adjusted to pH=3 with 6 N HCl aqueous solution,extracted with DCM:MeOH=10:1 (5.00 L*2), the combined organic layer waswashed with brine (2.00 L), dried over Na₂SO₄, filtered and concentratedunder vacuum to give a residue. Then the residue was diluted with MeCN(5.00 L), concentrated under vacuum, repeat this procedure twice toremove water to give TRIS-PEG2-CBZ (1.25 kg, 1.91 mol, 78.1% yield,95.8% purity) as light yellow oil. ¹HNMR: 400 MHz, MeOD, δ 7.30-7.35(5H), 5.07 (s, 2H), 3.65-3.70 (m, 16H), 3.59 (s, 4H), 3.45 (t, J=5.6Hz), 2.51 (t, J=6.0 Hz), 2.43 (t, 6.4 Hz).

General Procedure for Preparation of Compound 3C

To a solution of Compound 1C (155 g, 245 mmol, 1.00 eq) in ACN (1500 mL)was added TBTU (260 g, 811 mmol, 3.30 eq), DIEA (209 g, 1.62 mol, 282mL, 6.60 eq) and Compound 2C (492 g, 811 mmol, 3.30 eq. TsOH) at 0° C.,the mixture was stirred at 15° C. for 16 hrs. LCMS showed the desired MSwas given. The mixture was concentrated under vacuum to give a residue,then the mixture was diluted with DCM (2000 mL), washed with 1 N HClaqueous solution (700 mL*2), then saturated NaHCO₃ aqueous solution (700mL*2) and concentrated under vacuum. The crude was purified by columnchromatography to give Compound 3C (304 g, 155 mmol, 63.1% yield, 96.0%purity) as a yellow solid.

General Procedure for Preparation of Compound 4C

Two batches solution of Compound 3C (55.0 g, 29.2 mmol, 1.00 eq) in MeOH(1600 mL) was added Pd/C (6.60 g, 19.1 mmol, 10.0% purity) and TFA (3.34g, 29.2 mmol, 2.17 mL, 1.00 eq), the mixture was degassed under vacuumand purged with H. The mixture was stirred under H₂ (15 psi) at 15° C.for 2 hours. LCMS showed the desired MS was given. The mixture wasfiltered and the filtrate was concentrated under vacuum to give Compound4C (106 g, 54.8 mmol, 93.7% yield, 96.2% purity, TFA) as a white solid.

General Procedure or Preparation of Compound 5C

Two batches in parallel. To a solution of EDCI (28.8 g, 150 mmol, 1.00eq) in DCM (125 mL) was added compound 4a (25.0 g, 150 mmol, 1.00 eq)dropwise at 0° C., then the mixture was added to compound 4 (25.0 g, 150mmol, 1.00 eq) in DCM (125 mL) at 0° C., then the mixture was stirred at25° C. for 1 hr. TLC (Petroleum ether:Ethyl acetate=3:1, R_(f)=0.45)showed the reactant was consumed and one new spot was formed. Thereaction mixture was diluted with DCM (100 mL) then washed withaq.NaHCO₃ (250 mL*1) and brine (250 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether:Ethylacetate=100:1 to 3:1), TLC (SiO₂, Petroleum ether:Ethyl acetate=3:1),R_(f)=0.45, then concentrated under reduced pressure to give a residue.Compound 5C (57.0 g, 176 mmol, 58.4% yield, 96.9% purity) was obtainedas colorless oil and confirmed ¹HNMR:EW33072-2-P1A, 400 MHz, DMSO

δ 9.21 (s, 1H), 7.07-7.09 (m, 2H), 6.67-6.70 (m, 2H), 3.02-3.04 (m, 2H),2.86-2.90 (m, 2H)

General Procedure for Preparation of Compound 6

To a mixture of compound 3 (79.0 g, 41.0 mmol, 96.4% purity, 1.00 eq,TFA) and compound 6C (14.2 g, 43.8 mmol, 96.9% purity, 1.07 eq) in DCM(800 mL) was added TEA (16.6 g, 164 mmol, 22.8 mL, 4.00 eq) dropwise at0° C., the mixture was stirred at 15° C. for 16 hrs. LCMS(EW33072-12-P1B, Rt=0.844 min) showed the desired mass was detected. Thereaction mixture was diluted with DCM (400 mL) and washed with aq.NaHCO₃(400 mL*1) and brine(400 mL*1), then the mixture was diluted with DCM(2.00 L) and washed with 0.7 M Na₂CO₃ (1000 mL*3) and brine(800 mL*3),dried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was used to next step directly withoutpurification. Compound 6 (80.0 g, crude) was obtained as white solid andconfirmed via ¹HNMR:EW 33072-12-P1A, 400 MHz, MeOD δ 7.02-7.04 (m, 2H),6.68-6.70 (m, 2H), 5.34-5.35 (s, 3H), 5.07-5.08 (d, J=4.00 Hz, 3H),4.62-4.64 (d, J=8.00 Hz, 3H), 3.71-4.16 (m, 16H), 3.31-3.70 (m, 44H),2.80-2.83 (m, 2H), 2.68 (m, 2H), 2.46-2.47 (m, 10H), 2.14 (s, 9H), 2.03(s, 9H), 1.94-1.95 (d, J=4.00 Hz, 18H).

General Procedure for Preparation of TriGNal-TRIS-Peg2-Phosph 8c

Two batches were synthesized in parallel. To a solution of compound 6C(40.0 g, 21.1 mmol, 1.00 eq in DCM (600 mL) was addeddiisopropylammonium tetrazolide (3.62 g, 21.1 mmol, 10) eq) and compound7c (6.37 g, 21.1 mmol, 6.71 mL, 1.00 eq) in DCM (8.00 mL) drop-wise, themixture was stirred at 30° C. for 1 hr, then added compound 7c (3.18 g,10.6 mmol, 3.35 mL, 0.50 eq) in DCM (8.00 mL) drop-wise, the mixture wasstirred at 30° C. for 30 mins, then added compound 7c (3.18 g, 10.6mmol, 3.35 mL, 0.50 eq) in DCM (8.00 mL) drop-wise, the mixture wasstirred at 30° C. for 1.5 hrs. LCMS (EW33072-17-PI C1, Rt=0.921 min)showed the desired MS+1 was detected. LCMS (EW33072-17-P1C2, Rt=0.919min) showed the desired MS+1 was detected. Two batches were combined forwork-up. The mixture was diluted with DCM (1.20 L), washed withsaturated NaHCO₃ aqueous solution (1.60 L*2), 3% DMF in H₂O (1.60 L*2),H₂O (1.60 L*3), brine (1.60 L), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, DCM:MeOH:TEA=100:3:2) TLC(SiO₂, DCM:MeOH=10:1, R_(f)=0.45), then concentrated under reducedpressure to give a residue. Compound 8C (76.0 g, 34.8 mmol, 82.5% yield,96.0% purity) was obtained as white solid and confirmed via¹HNMR:EW33072-19-PIC, 400 MHz, MeOD δ 7.13-7.15 (d., =8.50 Hz, 2H),6.95-6.97 (dd, J=8.38, 1.13 Hz, 2H), 5.34 (d., =2.88 Hz, 3H), 0.09 (dd,J=11.26, 3.38 Hz, 3H), 4.64 (d, J=8.50 Hz, 3H), 3.99-4.20 (m, 12H),3.88-3.98 (m, 5H), 3.66-3.83 (m, 20H), 3.51-3.65 (m, 17H), 3.33-3.50 (m,9H), 2.87 (t, J=7.63 Hz, 2H), 2.76 (t, J=5.94 Hz, 2H), 2.42-2.50 (m,10H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94-1.95 (d, J=6.13 Hz, 18H),1.24-1.26 (d, J=6.75 Hz, 6H), 1.18-1.20 (d, J=6.75 Hz, 6H)

Example 7: In Vitro Hepatocyte Targeting Using GalNAc-Conjugated siRNAsand ASOs

In this experiment, a hepatocyte cell line expressing asialoglycoproteinreceptors and GFP will be treated with anti-GFP siRNAs or ASOsconjugated to a GalNAc moiety compared to a control experiment where thehepatocyte cell line is treated with anti-GFP siRNAs or ASOs that arenot conjugated to the GalNAc moiety. GFP mRNA and protein expression aremeasured, and the amount of GFP mRNA or protein expression in cellstreated with the GalNAc-conjugated siRNAs or ASOs is normalized andcompared to the amount of GFP mRNA or protein expression in cellstreated with the siRNAs or ASOs that are not GalNAc-conjugated. This mayallow for a determination of the hepatocyte targeting ability of theGalNAc moiety. Multiple GalNAc moieties may be conjugated to the siRNAsor ASOs and compared to see which GalNAc moiety results in optimalhepatocyte targeting. The GalNAc moieties to be tested in theseexperiments may a GalNAc moiety described herein.

Similar experiments may be performed in primary hepatocytes treated withthe siRNAs or ASOs conjugated or not to a GalNAc moiety, and a targetmRNA or target protein other than GFP may be assessed in the primaryhepatocytes.

Example 8: In Vivo Hepatocyte Targeting Using GalNAc-Conjugated siRNAsand ASOs

In this experiment, siRNAs or ASOs targeting a target mRNA will beconjugated to a GalNAc moiety and administered to mice (n=5/group), andcompared to a control experiment where the mice are administered siRNAsor ASOs without GalNAc conjugation. Mice are sacrificed 2 days later,and livers are frozen, later homogenized, and tested for target mRNA andprotein expression. The amount of target mRNA or protein expression inthe livers of mice treated with the GalNAc-conjugated siRNAs or ASOs isnormalized and compared to the amount of GFP mRNA or protein expressionin the livers of mice treated with the siRNAs or ASOs that are notGalNAc-conjugated. This may allow for a determination of the livertargeting ability of the GalNAc moiety. Multiple GalNAc moieties may beconjugated to the siRNAs or ASOs and compared to see which GalNAc moietyresults in optimal liver targeting. The GalNAc moieties included in thisexperiment may be those that exhibit the greatest degree of hepatocytetargeting. The GalNAc moieties to be tested in these experiments may aGalNAc moiety described herein.

Example 9: Inhibition of a Target mRNA in a Mouse Model of a LiverDisease Using GalNAc-Conjugated siRNAs and ASOs

In this experiment, a murine model of a liver disease (in this case,fatty liver disease) will be used to evaluate the effect of siRNA or ASOinhibition of a target mRNA. The target mRNA may encode any targetprotein where overexpression or overactivation plays a pathological rolein the liver disease. In the murine model, fatty liver disease isinduced by feeding mice a Western Diet (WD) containing 21.1% fat, 41%Sucrose, and 1.25% Cholesterol by weight (Teklad diets. TD. 120528) anda high sugar solution (23.1 g/L d-fructose (Sigma-Aldrich, G8270) and18.9 g/L d-glucose (Sigma-Aldrich, F0127)) for 12 weeks. At 4-week-oldC57BL/6J mice are fed a Western Diet instead of regular chow for 12weeks. The GalNAc moieties to be used in this experiments may a GalNAcmoiety described herein.

Briefly, mice are divided into five groups: Group 1: a fatty liverdisease group treated with non-targeting control siRNA, Group 2: a fattyliver disease group treated with non-targeting control ASO, Group 3: afatty liver disease group treated with an siRNA targeting a target mRNA,Group 4: a fatty liver disease group treated with an ASO targeting atarget mRNA, Group 5: control mice on a normal chow diet. Each groupcontains eight mice (4 males, 4 females). The siRNAs and ASOs of Groups1-4 each include a GalNAc moiety attached to the siRNA or ASO.

At weeks 12 weeks of Western Diet, blood samples are to be collectedfrom each group prior first treatment.

Administration of siRNA or ASO is achieved with a 200 μL subcutaneousinjection of naked siRNA or ASO resuspended in PBS at concentration of10 μM. On Study Day 0, Group 1 mice will be injected subcutaneously withnon-targeting control siRNA, Group 2 mice will be injectedsubcutaneously with non-targeting control ASO, Group 3 mice will beinjected subcutaneously with siRNA 1 targeting the target mRNA in amouse, Group 4 mice will be injected subcutaneously with ASO1 targetingthe target mRNA in a mouse, and Group 5 mice will be injectedsubcutaneously with vehicle. Every other week thereafter starting on Day14 the animals from each group will be dosed as on Day 0 for a total of5 injections.

Weekly blood draws will be taken and serum and plasma isolated. SerumALT, AST, total cholesterol and triglyceride levels are measured usingVITROS 5,1 FS (Ortho Clinical Diagnostics). Non-fasting plasma insulinis measured with the Ultrasensitive Mouse Insulin ELISA kit (CrystalChem, 90080) according to the manufacturer's instructions. Non-fastingblood glucose is assayed with the One Touch Ultra (Life Scan). HOMA IRand QUICKI will be calculated.

At the end of 12 weeks of Western Diet and siRNA/ASO treatment, mice areto be sacrificed by cervical dislocation following an intraperitonealinjection of 0.3 ml Nembutal (5 mg/ml). Terminal serum draw is collectedvia cardiac puncture and final serum ALT, AST, total cholesterol andtriglyceride levels are measure along with non-fasting plasma insulinand glucose. Livers are removed and divided into three sections; onesection placed in RNAlater for mRNA isolation, one section flash-frozenfor protein isolation, one section fixed in formalin and thenparaffin-embedded.

mRNA is isolated from tissue placed in RNAlater solution using thePureLink kit according to the manufacturer's protocol (ThermoFisher Cat.No. 12183020). The reverse transcriptase reaction is performed accordingto the manufacturer's protocol. Samples are stored at −80° C. untilreal-time qPCR is performed in triplicate using TaqMan Gene ExpressionAssays (Applied Biosystems FAM-probes using a BioRad iCycler). Adecrease in target mRNA expression in the liver tissue from mice isdosed with the siRNAs and ASOs compared to target mRNA levels in theliver tissue from mice is dosed with the non-specific control siRNA andASO. There is an expected decrease in the amount of SDF-1 in the livertissue from mice that receive the siRNAs and ASOs compared to the amountof SDF-1 in the liver tissue from mice that receive the non-specificcontrol siRNA or ASO. These results show that the siRNAs and ASOs elicitknockdown of the target mRNA and target protein in liver tissue, andthat the decrease in target mRNA and target protein expression iscorrelated with a decrease in SDF-1 production.

Formalin-fixed, paraffin-embedded liver sections are stained withhematoxylin and eosin (H&E) for assessment of liver histology, withSirius Red (Sigma, 365548-5G)/Fast Green (Sigma, F258) for assessment offibrosis, and with periodic acid-Schiff (PAS) for assessment of glycogenaccumulation. NAFLD Activity Score (NAS) and fibrosis stage areevaluated by an expert pathologist according to the NASH CRN scoringsystem13. The histological scoring is performed blinded, with noknowledge by the pathologist of the treatment(s) received. These resultsshow that the siRNAs and ASOs elicit knockdown of the target mRNA andtarget protein in liver tissue, and that the decrease in expression ofthe target mRNA and target protein is correlated with a decrease in NASand NASH CRN.

Example 10: Inhibition of a Mouse Model of a Liver Disease

In this experiment, a murine model of a liver disease (in this case,hypertriglyceridemia) will be used to evaluate the effect of siRNA orASO inhibition of a target protein expressed in the liver compared to ananti-mouse target protein antibody. The mouse strain C57Bl/6 Apoetm1Uncmice will be maintained on a high fat Western diet (Research Diets,D12492: 60% fat by calories). The target protein may be any targetprotein where overexpression or overactivation plays a pathological rolein the liver disease. The GalNAc moieties to be used in this experimentsmay a GalNAc moiety described herein.

Four groups of mice (n=16/group) will be utilized in this study. Animalswill be maintained on a high fat diet during the study. On Day −4 beforethe first injection, chow will be removed for an overnight fast. On Day−3 before the first injection, all animals will be anesthetized and 300μL of blood collected in serum separator tubes via the submandibularvein to assess baseline triglyceride, serum glucose, insulinsensitivity, total cholesterol levels, HDL Cholesterol levels, liverfunction and serum levels of target protein. On Study Day 0, Group 1mice will be injected intraperitoneally with 600 μL normal saline, Group2 mice will be injected intraperitoneally with 600 μg of anti-mousetarget protein antibody in 600 μL, Group 3 mice will be injectedsubcutaneously with 150 μg of GalNAc-siRNA targeting an mRNA encodingthe target protein in a mouse in 200 μL of normal saline, and Group 4mice will be injected subcutaneously with 150 μg of GalNAc-ASO targetingthe mRNA encoding the target protein in 200 μL of normal saline. On theafternoon of Day 3, the chow will be removed from all Groups for anovernight fast. On Day 4, the animals from all Groups will beanesthetized and 150 μL of blood collected in serum separator tubes viathe submandibular vein to assess serum triglycerides, glucose, totalcholesterol, HDL cholesterol and levels of the target protein. Animalsfrom all groups will then undergo an oral glucose tolerance test andinsulin tolerance test to evaluate insulin sensitivity. Chow will besupplied again as normal after blood has been collected and insulinsensitivity tests conducted. Weekly thereafter starting on Day 7 theanimals from Group 2 will be dosed as on Day 0 for a total of 15injections. Every other week thereafter starting on Day 14 the animalsfrom Group 3 and Group 4 will be dosed as on Day 0 for a total of 8injections. Every other week starting on Day 10, the mice from allGroups will be fasted (overnight) and bled (150 μL into serum separatortubes) to assess serum triglyceride, glucose, total cholesterol, HDLcholesterol and levels of target protein, and undergo insulinsensitivity tests. On the third day after the final injection, the chowwill be removed from all Groups for an overnight fast. On the fourth dayafter the final injection, the animals from all Groups will beanesthetized, euthanized and bled via cardiac puncture to collect 500 μLof blood into serum separator tubes to assess triglyceride, serumglucose, insulin sensitivity, total cholesterol levels, HDL cholesterollevels, liver function and serum levels of target protein. Tissue fromthe liver, small intestine and mesenteric lymph nodes will be collectedfrom all animals and immersed in 10% neutral buffered formalin forhistopathological analysis. A liver sample will also be collected fromall animals and placed in RNAlater. The levels of target mRNA will beassessed by RT-qPCR using TaqMan assays for the mouse target protein andthe mouse housekeeping gene PPIA.

Animals treated with the antibody (Group 2), mice treated with theGalNAc-siRNA (Group 3), and mice treated with the GalNAc-ASO (Group 4)are expected to have decreased triglycerides, total serum cholesterol,serum glucose as well as decreased serum target protein levels, andincreased HDL cholesterol and insulin sensitivity, compared with micefrom Group 1 (saline). Animals in Group 2 and Group 3 are also expectedto have decreased target mRNA in liver samples.

Example 11: Inhibition of a Target mRNA in Non-Human Primates UsingGalNAc-siRNA and GalNAc-ASO

In this experiment, a NHP model of hypertriglyceridemia is used toevaluate the effect of siRNA or ASO inhibition of the target mRNAexpressed in the liver. The target protein may be any target proteinwhere overexpression or overactivation plays a pathological role in theliver disease. Three groups of cynomolgus monkeys will be used(n=5/group) that are placed on a high-fat diet (Western Primate Diet,5S2T) before the initiation of the study. Alternatively, three groups ofrhesus monkeys will be used (n=5/group) that are placed on a highfructose diet before the initiation of the study. Animals are to begiven 7 biweekly subcutaneous injections of saline (Group 1),GalNAc-siRNA (Group 2), or GalNAc-ASO (Group 3). The modifiedGalNAc-siRNA sequences may include any modification pattern describedherein. The GalNAc moieties to be used in this experiments may a GalNAcmoiety described herein. Blood samples for lipid and glycemicmeasurements will be collected at baseline and at 4, 8, and 14 weeks ofthe study and analyzed for lipid content, serum glucose, insulinsensitivity and target protein. All animals from each group arenecropsied 2 weeks after the last blood collection. Tissue from theliver, small intestine and mesenteric lymph nodes will be collected fromall animals and immersed in 10% neutral buffered formalin forhistopathological analysis. A liver sample will also be collected fromall animals and placed in RNAlater. The levels of target mRNA will beassessed by RT-qPCR using TaqMan assays for cynomolgus or rhesus targetprotein and the cynomolgus or rhesus housekeeping gene PPIA.

It is expected that animals treated with the GalNAc-siRNA (Group 2) andanimals treated with the GalNAc-ASO (Group 3) will show decreasedtriglycerides, total serum cholesterol and serum glucose as well asdecreased serum target protein levels, and increased HDL cholesterol andinsulin sensitivity, compared with animals from Group 1 (saline). It isalso expected that animals in Group 1 and Group 3 will show decreasedtarget mRNA in liver samples.

Example 12: Inhibition of a Target mRNA in a Clinical Trial UsingGalNAc-siRNA and GalNAc-ASO

In this study, human subjects with hypertriglyceridemia are used toevaluate the effect of siRNA or ASO inhibition of a target mRNAexpressed in the liver. The target protein may be any target proteinwhere overexpression or overactivation plays a pathological role in theliver disease. Selection criteria for inclusion in the study are ages40-90, BMI ≥30, and serum triglycerides ≥250 mg/dL. Three groups ofsubjects will be included (n=15/group) in the study. Subjects are to begiven 5 weekly subcutaneous injections of saline (Group 1), GalNAc-siRNA(Group 2), or GalNAc-ASO (Group 3). The GalNAc moieties to be used inthese experiments may include a GalNAc moiety described herein.

The siRNA or ASO sequences are to be from a selection set that showshigh activity in cells in culture or in experiments describe in theother examples. Blood samples for lipid and glycemic measurements willbe collected at baseline and at 3, 6, and 12 weeks of the study andanalyzed for lipid content, serum glucose, insulin sensitivity, targetprotein, and liver and kidney function.

It is expected that subjects treated with the GalNAc-siRNA (Group 2) andsubjects treated with GalNAc-ASO (Group 3) will show decreasedtriglycerides, total serum cholesterol and serum glucose as well asdecreased serum target protein levels, and increased HDL cholesterol andinsulin sensitivity, compared with subjects from Group 1 (saline).

Example 13: Oligonucleotide Synthesis

RNAi agents (e.g. siRNAs) were synthesized according to phosphoramiditetechnology on a solid phase used in oligonucleotide synthesis. A K&Aoligonucleotide synthesizer was used. Syntheses were performed on asolid support made of controlled pore glass (CPG, 500 Å or 600 Å,obtained from AM Chemicals, Oceanside, CA, USA). All 2′-OMe and 2′-Fphosphoramidites were purchased from Hongene Biotech (Union City, CA,USA). All phosphoramidites were dissolved in anhydrous acetonitrile (100mM) and molecular sieves (3 Å) were added. 5-Benzylthio-1H-tetrazole(BTI, 250 mM in acetonitrile) or 5-Ethylthio-1H-tetrazole (ETT, 250 mMin acetonitrile) was used as activator solution. Coupling times were9-18 min (EmpGalNAc), 6 min (2′OMe and 2′F). In order to introducephosphorothioate linkages, a 100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous acetonitrile was employed.

After solid phase synthesis, the dried solid support was treated with a1:1 volume solution of 40 wt. % methylamine in water and 28% ammoniumhydroxide solution (Aldrich) for two hours at 30° C. The solution wasevaporated and the solid residue was reconstituted in water and purifiedby anionic exchange HPLC using a TKSgel SuperQ-5PW 13u column. Buffer Awas 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile andbuffer B was the same as buffer A with the addition of 1 M sodiumchloride. UV traces at 260 nm were recorded. Appropriate fractions werepooled then desalted using Sephadex G-25 medium.

Equimolar amounts of sense and antisense strand were combined to preparea duplex. The duplex solution was prepared in 0.1×PBS(Phosphate-Buffered Saline, 1×, Gibeo). The duplex solution was annealedat 95° C. for 5 min, and cooled to room temperature slowly. Duplexconcentration was determined by measuring the solution absorbance on aUV-Vis spectrometer at 260 nm in 0.1×PBS. For some experiments, aconversion factor was calculated from an experimentally determinedextinction coefficient.

Example 14: In Vivo Hepatocyte Targeting Using GalNAc-Conjugated siRNAsand ASOs

In this experiment, siRNAs or ASOs targeting a target mRNA will beconjugated to a GalNAc moiety and administered to mice (n=5/group), andcompared to a control experiment where the mice are administered siRNAsor ASOs without GalNAc conjugation. Mice are sacrificed 2 days later,and livers are frozen, later homogenized, and tested for target mRNA andprotein expression. The amount of target mRNA or protein expression inthe livers of mice treated with the GalNAc-conjugated siRNAs or ASOs isnormalized and compared to the amount of GFP mRNA or protein expressionin the livers of mice treated with the siRNAs or ASOs that are notGalNAc-conjugated. This may allow for a determination of the livertargeting ability of the GalNAc moiety. Multiple GalNAc moieties may beconjugated to the siRNAs or ASOs and compared to see which GalNAc moietyresults in optimal liver targeting. The GalNAc moieties may be thosethat exhibit the greatest degree of hepatocyte targeting in Example 6.The GalNAc moieties to be tested in these experiments may a GalNAcmoiety described herein such as Compound 1 or Compound 2.

Example 15: Knockdown of PLIN1 in Mice by GalNAc-Conjugated siRNAs

An example GalNAc Moiety, ETL17, was conjugated to siRNAs targeting anexample target mRNA. Sequences are shown in Table 2. In the table, Nf(e.g. Af, Cf, Gf, Tf, or Uf) is a 2′ fluoro-modified nucleoside, n (e.g.a, c, g, t, or u) is a 2′ O-methyl modified nucleoside, and “s” is aphosphorothioate linkage. The siRNAs were tested for activity in mice.

Six to eight week old female mice (C57Bl/6) were injected with 10 uL ofa recombinant adeno-associated virus 8 (AAV8) vector (1.7×10E¹³ genomecopies/mL) by the retroorbital route on Day −14. The recombinant AAV8contained the sequence of human PLIN1 (NM_002666.5) under the control ofthe human thyroxine binding globulin promoter in an AAV2 backbonepackaged in AAV8 capsid (AAV8-TBG-h-PLIN1). On Day 0, infected mice(n=4) were given a subcutaneous injection of a single 100 ug dose of aGalNAc-conjugated siRNA or PBS as vehicle control.

Mice were euthanized on Day 10 after subcutaneous injection and a liversample from each was collected and placed in RNAlater (ThermoFisherCatalog #AM7020) until processing. Total liver RNA was prepared byhomogenizing the liver tissue in homogenization buffer (Maxwell RSCsimplyRNA Tissue Kit) using a Percellys 24 tissue homogenizer (BertinInstruments) set at 5000 rpm for two 10 second cycles. Total RNA fromthe lysate was purified on a Maxwell RSC 48 platform (PromegaCorporation) according to the manufacturer's recommendations.Preparation of cDNA was performed using Quanta qScript cDNA SuperMix(VWR, Catalog #95048-500) according to the manufacturer's instructions.The relative levels of liver PLIN1 mRNA were assessed by RT-qPCR intriplicate on a QuantStudio™ 6 Pro Real-Time PCR System using TaqManassays for human PLIN1 (ThermoFisher, assay #Hs01106925_m1) and themouse housekeeping gene PPIA (ThermoFisher, assay #Mm02342430_g1) andPerfeCTat qPCR FastMix®, Low ROX™ (VWR, Catalog #101419-222). Data werenormalized to the mean PLIN1 mRNA level in animals receiving PBS.Results are shown in Table 3. All of the siRNAs tested caused areduction in mean liver PLIN1 mRNA on Day 10 relative to mice receivingPBS. These data indicate that siRNAs conjugated to a GalNAc moiety suchas ETL17 are useful for knocking down a target mRNA in the liver.

TABLE 2 Example siRNA Sequences siRNA SEQSense Strand Sequence (5′-3′) with SEQ Name ID NO GalNAc moiety ID NOAntisense Strand Sequence (5′-3′) ETD01899 1[ETL17]scaguuuuuAfaGfggacaccasusu  8 usGfsgUfgUfcCfcUfuAfaAfaAfcUfgsusuETD01900 2 [ETL17]suuuuuAfAfGfGfgAfcaccagaasusu  9usUfscUfgGfuGfuCfcCfuUfaAfaAfasusu ETD01901 3[ETL17]suuuugaCfaCfaUfucuuagcasusu 10 usGfscUfaAfgAfaUfgUfgUfcAfaAfasusuETD01902 4 [ETL17]suugaCfaCfaUfuCfuuagcacasusu 11usGfsuGfcUfaAfgAfaUfgUfgUfcAfasusu ETD01903 5[ETL17]sacauucuuAfGfcacugaacasusu 12 usGfsuUfcAfgUfgCfuAfaGfaAfuGfususuETD01904 6 [ETL17]sugcaUfagUfCfaCfucuuuugasusu 13usCfsaAfaAfgAfgUfgAfcUfaUfgCfasusu ETD01905 7[ETL17]saacuaCfUfgCfaUfaauauggasusu 14usCfscAfuAfuUfaUfgCfaGfuAfgUfususu

TABLE 3 Relative human PLINI mRNA Levels in Livers of Mice Dose MeanPLIN1 mRNA (Normalized Group n Treatment (ug) to Group 1, Day 10) 1 4PBS  0 1.00 2 4 ETD01899 100 0.79 3 4 ETD01900 100 0.30 4 4 ETD01901 1000.37 5 4 ETD01902 100 0.24 6 4 ETD01903 100 0.83 7 4 ETD01904 100 0.58

Example 16: Knockdown of MST1 in Mice by GalNAc-Conjugated siRNAs

ETL17 was conjugated to siRNAs targeting another example target mRNA.The siRNAs were attached to the GalNAc ligand ETL17 followed by aphosphorothioate linkage at the 5′ end of the sense strand. The siRNAsare described in Table 6. In the table, Nf (e.g. Af, Cf. Gf. Tf, or Uf)is a 2′ fluoro-modified nucleoside, dN (e.g. dA, dC, dG, dT, or dU) is a2′ deoxy-modified nucleoside, n (e.g. a, c, g, t, or u) is a 2′ O-methylmodified nucleoside, and “s” is a phosphorothioate linkage.

Six to eight week old female mice (C57Bl/6) were injected with 5 uL of arecombinant adeno-associated virus 8 (AAV8) vector (2.7×10E13 genomecopies/mL) by the retroorbital route. The recombinant AAV8 contained theopen reading frame and the majority of the 3′UTR of the human MST1sequence (NM_020998.4) under the control of the human thyroxine bindingglobulin promoter in an AAV2 backbone packaged in AAV8 capsid(AAV8-TBG-h-MST1). On Day 13 after infection, serum was collected andthe level of human MSP in each mouse was measured using the HumanMSP/MST1 DuoSet ELISA from R&D (Catalog #DY352). The manufacturer'sinstructions regarding all reagent preparations for buffers andsolutions was followed. A serum sample dilution of 1:50 was utilized forall test samples. Recombinant MSP included in the kit was used to createa standard curve of 10,000 pg/mL to 0 pg/mL. The optical density of theplate was read at 450 nm using a PerkinElmer Envision multimode platereader. The concentration of MSP in each mouse serum sample wascalculated from the standard curve by interpolation using least squaresfit (Prism version 9, Software MacKiev).

Mice were allocated into groups (n=3) such that the groups had similarserum levels of MSP and then given a subcutaneous injection of a single60 ug dose of a GalNAc-conjugated siRNA or PBS as vehicle control. OnDays 0, 4 and 12 after injection, serum was collected to assess serumMSP concentrations by ELISA using the methods described above. The MSPserum concentration at each timepoint was made relative to the level ofMSP in the Day 0 sample for each individual mouse. The results are shownin Table 4. These data indicate that siRNAs conjugated to a GalNAcmoiety such as ETL17 are useful for knocking down proteins secreted byan additional target mRNA in the liver.

Mice were sacrificed on Day 12 and a liver sample from each wascollected and placed in RNAlater (ThermoFisher Cat #AM7020) untilprocessing. Total liver RNA was prepared by homogenizing the livertissue in homogenization buffer (Maxwell RSC simplyRNA Tissue Kit) usinga Percellys 24 tissue homogenizer (Bertin Instruments) set at 5000 rpmfor two 10 second cycles. Total RNA from the lysate was purified on aMaxwell RSC 48 platform (Promega Corporation) according to themanufacturer's recommendations. Preparation of cDNA was performed usingQuanta qScript cDNA SuperMix (VWR, Catalog #95048-500) according to themanufacturer's instructions. The relative levels of liver MST1 mRNA wereassessed by RT-qPCR in triplicate on a QuantStudio™ 6 Pro Real-Time PCRSystem using TaqMan assays for human MST1 (ThermoFisher, assay#Hs00360684_m1) and the mouse housekeeping gene PPIA (ThermoFisher,assay #Mm02342430_g1) and PerfeCTa® qPCR FastMix®, Low ROX™ (VWR,Catalog #101419-222). Data were normalized to the level in animalsreceiving PBS. Results are shown in Table 5. These data indicated thatsiRNAs conjugated to a GalNAc moiety such as ETL17 are useful forknocking down the additional target mRNA in the liver

TABLE 4 Relative Mean Serum Human MSP Levels in AAV8-TBG-h-MST1 MiceMean serum human MSP Dose (Relative to Day 0) Group n Treatment (ug) Day0 Day 4 Day 12 1 3 PBS 1.00 1.12 1.54 2 3 ETD01867 60 1.00 0.35 0.24 3 3ETD01963 60 1.00 0.46 0.42 4 3 ETD01964 60 1.00 0.35 0.15 5 3 ETD0196560 1.00 0.32 0.26 6 3 ETD01966 60 1.00 0.30 0.16 7 3 ETD01868 60 1.000.67 ND 8 3 ETD01967 60 1.00 0.41 0.27 9 3 ETD01968 60 1.00 0.53 0.30 103 ETD01969 60 1.00 0.68 0.45 11 3 ETD01970 60 1.00 0.51 0.59 12 3ETD01971 60 1.00 0.60 0.42 13 3 ETD01972 60 1.00 0.24 0.17 ND, notdetermined

TABLE 5 Relative Human MST1 mRNA Levels in Livers of AAV8-TBG-h-MST1Mice Dose Mean human MST1 mRNA Group n Treatment (ug) (Relative to Group1, Day 12) 1 3 PBS 1.00 2 3 ETD01867 60 0.40 3 3 ETD01963 60 0.76 4 3ETD01964 60 0.60 5 3 ETD01965 60 0.25 6 3 ETD01966 60 0.33 7 3 ETD0186860 0.28 8 3 ETD01967 60 0.07 9 3 ETD01968 60 0.32 10 3 ETD01969 60 0.1511 3 ETD01970 60 0.24 12 3 ETD01971 60 0.31 13 3 ETD01972 60 0.08

TABLE 6 siRNAs Screened for Activity in AAV8-TBG-h-MST1 Mice siRNA SEQSense Strand Sequence SEQ Name ID NO (5′-3′) with GalNAc Moiety ID NO.Antisense Strand Sequence (5′-3′) ETD01867 43 [ETL17]sucuuGfucAfGfacauaa54 usGfscUfuUfaUfgUfcUfgAfcAfaGfasusu agcasusu ETD01963 44[ETL17]sucuuGfucAfGfacauaa 55 usGfscuuUfaUfgUfcUfgAfcAfaGfasusu agcasusuETD01964 45 [ETL17]sucuuGfucAfGfacauaa 56usGfscuuUfaugUfcUfgAfcAfaGfasusu agcasusu ETD01965 46[ETL17]sucuuGfucAfGfacauaa 57 usGfscUfuUfaUfgucUfgAfcAfaGfasusu agcasusuETD01966 47 [ETL17|sucuuGfucAfGfacauaa 58usGfscUfuuAfugUfcUfgAfcAfaGfasusu agcasusu ETD01967 48[ETL17]suuguCfadGaCfaUfaaa 59 usUfsgGfcUfuUfaUfgUfcUfgAfcAfasusugccaasusu ETD01968 49 [ETL17]suugucagaCfdAUfaaag 60usUfsgGfcUfuUfaUfgUfcUfgAfcAfasusu ccaasusu ETD01969 50[ETL17]suuguCfagaCfaUfaaag 61 usUfsggcUfuUfaUfgUfcUfgAfcAfasusu ccaasusuETD01970 51 [ETL17]suuguCfagaCfaUfaaag 62usUfsgGfcUfuUfaugUfcUfgAfcAfasusu ccaasusu ETD01971 52[ETL17]suuguCfagaCfaUfaaag 63 usUfsggcUfuUfaugUfcUfgAfcAfasusu ccaasusuETD01972 53 [ETL17]suuguCfagaCfaUfaaag 64usUfsggCfuuuaUfgUfcUfgAfcAfasusu ccaasusu

Example 17: Knockdown of an Additional Target mRNA in Mice byGalNAc-Conjugated siRNAs

Three groups (n=4/group) of 8-week-old male ICR mice (Invigo) wereutilized in a study. On Study Day 0, Group 1 mice were injectedsubcutaneously with 100 uL of sterile PBS, Group 2 mice weresubcutaneously injected with 60 ug of siRNA 1811 in 100 uL of sterilePBS, and Group 3 mice were subcutaneously injected with 200 ug siRNA1818 in 100 uL of sterile PBS. On Study Day 14, the animals from allGroups were anesthetized and then euthanized. A liver sample wascollected from all animals and placed in RNAlater™ StabilizationSolution (Thermo Fisher, Catalog #AM7020). The liver samples wereprocessed in homogenization buffer (Maxwell RSC simplyRNA Tissue Kit)using Soft Tissue Homogenizing Kit CK14 (Bertin Instruments, catalog#P000933-LYSK0-A) in a Percellys 24 tissue homogenizer (BertinInstruments) set at 5000 rpm for two 10 second cycles. Total RNA fromthe liver lysate was purified on a Maxwell RSC 48 platform (PromegaCorporation) according to the manufacturer's recommendations. Therelative level of Gene A mRNA in each liver sample was assessed byRT-qPCR on a QuantStudio 6 Pro instrument (Applied Biosystems) usingTaqMan assays for mouse Gene A and the mouse housekeeping gene PPIA, andthen normalized to the mean value of the control mice (Group 1) usingthe delta-delta Ct method.

In this example, an additional GalNAc moiety (ETL1) was compared toETL17. The results of the liver mRNA analyses are shown in Table 7.Animals treated with ETL1-targeted siRNA (ETD01811, Group 2) had 78%relative knockdown while ETL17-targeted siRNA (siRNA 1818, Group 3) had83% knockdown of liver Gene A mRNA levels, compared with mice injectedwith PBS (Group 1). Thus, a compound having a GalNAc moiety describedherein may be superior or at least as effective relative to anotherGalNAc moiety in knocking down a target such as a target mRNA.

TABLE 7 Day 14 Gene A mRNA liver levels in mice treated with siRNAstargeting Gene A Group # Treatment Mean 1 PBS 1.00 2 siRNA 1811 (withcontrol GalNAc ETL1) 0.22 3 siRNA 1818 (with ETL17) 0.17

ETL1 is shown below, in which J indicates an attachment to anoligonucleotide:

Example 18. Testing the Activity of MST1 siRNAs ETD01821, ETD01822,ETD01823 and ETD01826 in Non-Human Primates

Four groups (n=3/group) of 3-6 kg male cynomolgus monkeys (ZhaoqingChuangyao Biotechnology Co., Ltd and Guangzhou Xianngguan BiotechnologyCo., Ltd) were utilized for this study.

On Study Day 0, Group 1 cynomolgus monkeys were injected subcutaneouslywith a single 5 mg/kg dose (0.2 mL dose volume/kg body weight) ofETD01821 at an siRNA concentration of 25 mg/mL formulated in PBS, Group2 cynomolgus monkeys were injected with a single 5 mg/kg dose (0.2 mLdose volume/kg body weight) of ETD01822 at an siRNA concentration of 25mg/mL formulated in PBS, Group 3 cynomolgus monkeys were injected with asingle 5 mg/kg dose (0.2 mL dose volume/kg body weight) of ETD01823 atan siRNA concentration of 25 mg/mL formulated in PBS, Group 4 cynomolgusmonkeys were injected with a single 5 mg/kg dose (0.2 mL dose volume/kgbody weight) of ETD01826 at an siRNA concentration of 25 mg/mLformulated in PBS, The siRNA sequences are shown in Table 8, where “Nf”is a 2′ fluoro-modified nucleoside, “n” is a 2′ O-methyl modifiednucleoside, and “s” is a phosphorothioate linkage. The injection wasgenerally well-tolerated as measured by clinical symptoms.

All cynomolgus monkeys had no abnormal clinical symptoms during theduration of the study except animal No. 101 which was found dead on Day65 post-dose. Necropsy revealed severe gastric perforation that may havebeen the cause of death. This can spontaneously occur in cynomolgusmonkeys.

On Study Days −8, −1, 7, 14, 21 and Day 28 body weights were recorded.Results are shown in Table 9. Data indicates that the animals in group 1to 4 did not have a meaningful change in their body weight.

On Study Days −8, −2, 7, 14, and Day 28 blood was collected into tubeswith no anti-coagulant and serum collected. Clinical chemistryparameters containing ALT, AST. ALP, TBIL, DBIL, GLU, GGT, TP, TG, CHOL,HDL, LDL, BUN and CREA were analyzed. Animals treated with ETD01821,ETD01822, ETD01823, or ETD01926 showed no meaningful change in ALT, AST,ALP, TBIL, DBIL, GL U, GGT, TP, TG, CHOL, HDL, LDL, BUN and CREAstarting on Study Day 7 though Study Day 28 when compared to Study Day−8 and Study Day −2, prior to treatment. The results from the clinicalchemistry indicated that all the siRNAs were generally well tolerated.Results are shown in Tables 10-14.

On Study Days −8, −2, 7, 14, and Day 28 about 1 mL of whole blood wascollected into tubes with EDTA-K2 as the anti-coagulant. Hematologyparameters including WBC, NEUT, LYMP, MONO, EOS, BASO, RBC, HGB, HCT,MCV, MCH, MCHC, RDW, PLT, MPV, PCT and PDW were analyzed. Animalstreated with ETD01821, ETD01822, ETD01823, or ETD01926 showed nomeaningful change in these hematological parameters starting on StudyDay 7 though Study Day 28 when compared to Study Day −8 and Study Day−2, prior to treatment. The results from the hematological analysesindicated that all the siRNAs were generally well tolerated. Results areshown in Tables 15-19.

On Study Days −8, −2, 7, 14, 28, 42, 56, 70, 77, 84, 91, 98 and Day 105,blood was collected into tubes with no anti-coagulant and serumcollected for determination of serum macrophage stimulating protein(MSP) levels. Additional serum samples were taken at later timepoints,namely on Days 42, 56, 70, 77, 84, 91, 98 and Day 105. A customAlphaLISA assay (PerkinElmer) was used to evaluate individual macrophagestimulating protein (MSP) concentrations in the monkey serum samples.Briefly, 5 uL of serum sample diluted 1:50 in 1× AlphaLISA HiBlock wasplaced into a well of a 96 well plate followed by addition of 5 uL of 4×anti-MSP acceptor bead solution. After incubation at room temperaturefor 30 minutes, 5 uL of 4× biotinylated anti-MSP antibody solution wasadded and the plate incubated at room temperature for 60 minutes. Next,5 uL of 4× streptavidin donor bead solution was added and the plateincubated for a further 30 minutes at room temperature. The plate wasanalyzed on an Envision 2105 Multimode Plate Reader (PerkinElmer). Astandard curve was generated using recombinant human MSP (R&D Systems).The MSP serum concentration for each individual at each timepoint wasmade relative to the mean of the MSP serum concentration for thatindividual on Days −2 and Day −8. Results for Group means are shown inTable 20 and individual values are shown in Table 21. Serum levels ofMSP were decreased in all animals after treatment with test articlesstarting at Day 7 and remained decreased at least through Day 28.Monkeys treated with ETD01821 had the greatest decrease in scrum MSPlevels relative to pre-dose levels, showing decreased mean serum MSPlevel compared to pre-dose levels through Day 105.

On Study Day −8 and Day 28, the animals were anesthetized with Zoletil(1.5-5.0 mg/kg, i.m.) and xylazine (0.5-2.0 mg/kg, i.m.) and 3-4 mgliver biopsy was collected. The biopsy was then placed in 10 v/vRNAlater in 20 seconds and stored for 24 hrs at 4° C., the RNAlater™Stabilization Solution (Thermo Fisher, Catalog #AM7020) was then removedand the liver tissue was stored in freezer until they were shipped toEmpirico. There were no abnormal clinical observations for all animalsafter liver biopsy collection on Day −2 or Day 28. The liver sampleswere processed in homogenization buffer (Maxwell RSC simplyRNA TissueKit) using Soft Tissue Homogenizing Kit CK14 (Bertin Instruments,catalog #P000933-LYSK0-A) in a Percellys 24 tissue homogenizer(BertinInstruments) set at 5000 rpm for two 10 second cycles. Total RNA fromthe liver lysate was purified on a Maxwell RSC 48 platform (PromegaCorporation) according to the manufacturer's recommendations.Preparation of cDNA was performed using Quanta qScript cDNA SuperMix(VWR, Catalog #95048-500) according to the manufacturers instructions.The relative levels of liver MST1 mRNA were assessed in biplexedreactions by RT-qPCR in triplicate using TaqMan assays for Macacafascicularis MST1 (ThermoFisher, assay #Mf01117426_g1) and the Macacafascicularis housekeeping gene GAPDH (ThermoFisher, assay #˜Mf04392546_g1) in PerfeCTa qPCR FastMix Reaction Mix (VWR). The sampleswere assessed on a QuantStudio™ 6 Pro Real-Time PCR System. Thedelta-delta Ct method was used to calculate relative amounts of MST1mRNA. Group mean relative MST1 mRNA levels relative to Day −8 are shownin Table 22. Consistent with the decrease in serum MSP levels asmeasured by AlphaLISA, treatment with 5 mg/kg of the test articlesETD01821, ETD01822, ETD01823 or ETD01826 resulted in a decrease in theliver levels of MST1 mRNA on Day 28 compared to the pre-dose Day −8levels.

TABLE 8 Example siRNA Sequences Sense Strand Antisense siRNA SEQSense Strand Sequence (5′-3′) Strand Name ID NO: with GalNAc moietySEQ ID NO: Antisense Strand Sequence (5′-3′) ETD01821 15[ETL17]sgguccuGfGfAfAfGfgaau 19 usAfsuAfaUfuCfcUfuCfcAfgGfaCfcsusuDauasusu ETD01822 16 [ETL17]sAfaCfuUfcUfudGuCfaga 20usUfsaUfgUfcUfgAfcAfaGfaAfgUfususu CfaUfaasusu ETD01823 17[ETL17]scuucUfUfgUfCfagacaua 21 usUfsuUfaUfgUfcUfgAfcAfaGfaAfgsusuaaasusu ETD01826 18 [ETL17]scaaccAfGfGfAfGfuguaa 22usAfsuGfuUfaCfaCfuCfcUfgGfuUfgsusu cauasusu

TABLE 9 Body Weight (kg) Treatment Animal Days prior to dose andpost-dose group No. Gender −8 −1 0 7 14 21 28 G1: ETD01821 101 Male 5.65.5 5.6 5.7 5.7 5.8 5.6 102 Male 6.0 6.0 6.0 5.9 6.1 6.2 6.1 103 Male4.5 4.6 4.5 4.6 4.6 4.7 4.5 G2: ETD01822 201 Male 6.5 6.5 6.5 6.6 6.56.5 6.5 202 Male 4.3 4.3 4.4 4.4 4.5 4.6 4.4 203 Male 5.5 5.5 5.6 5.65.6 5.4 5.6 G3: ETD01823 301 Male 4.7 4.5 4.6 4.7 4.8 4.7 4.9 302 Male4.6 4.6 4.6 4.6 4.6 4.7 4.5 303 Male 3.8 3.8 3.7 3.8 3.7 3.6 3.7 G4:ETD01826 401 Male 3.7 3.6 3.7 3.8 3.7 3.8 3.7 402 Male 5.9 5.9 6.0 5.96.0 6.1 6.0 403 Male 4.5 4.6 4.6 4.6 4.5 4.5 4.6

TABLE 10 Individual and Mean Clinical Chemistry Parameters Results onPre-dose (Day −8) Treatment group G1: ETD01821 G2: ETD01822 Animal No.101 102 103 Mean SD 201 Gender Male Male Male Male Animal ID SC1702037SC1509029 175151C SC1508015 Parameters ALT (U/L) 27.5 15.2 27.0 23.26.96 15.0 (unit) AST (U/L) 29.9 22.8 29.6 27.4 4.02 20.6 ALP (U/L) 530228↓   667 475 224 209↓   TBIL (μmol/L) 1.54  1.37 1.28 1.40 0.13  1.04DBIL (μmol/L) 0.11  0.53 0.46 0.37 0.23  0.17 GLU (mmol/L) 3.92  3.383.03 3.44 0.45  2.88 GGT (U/L) 102 76.2 91.4 89.8 12.8 71.0 TP (g/L)73.4 68.2 70.2 70.6 2.60 64.4 TG (mmol/L) 0.96  0.37 0.78 0.70 0.30 0.31 BUN (mmol/L) 14.3 10.9 13.6 13.0 1.78 17.4 CREA (μmol/L) 75.2 64.971.5 70.5 5.22 76.9 Treatment group G2: ETD01822 Animal No. 202 203 MeanSD Gender Male Male Animal ID SC1704115 SC1703011 Parameters ALT (U/L)29.0 31.4 25.1 8.86 (unit) AST (U/L) 42.3 29.2 30.7 10.9 ALP (U/L) 473675 453 234 TBIL (μmol/L) 1.68 2.06 1.59 0.52 DBIL (μmol/L) 0.38 0.360.30 0.12 GLU (mmol/L) 2.92 3.54 3.11 0.37 GGT (U/L) 69.4 64.2 68.2 3.57TP (g/L) 69.1 67.8 67.1 2.40 TG (mmol/L) 0.22 0.55 0.36 0.17 BUN(mmol/L) 10.7 15.3 14.5 3.44 CREA (μmol/L) 67.2 76.7 73.6 5.54 Treatmentgroup G3: ETD01823 G4: ETD01826 Animal No. 301 302 303 Mean SD 401Gender Male Male Male Male Animal ID 177695C SC1704077 176313C SC1708089Parameters ALT (U/L) 41.2 35.4 26.5 34.4 7.40 19.6 (unit) AST (U/L) 33.432.5 29.2 31.7 2.21 28.2 ALP (U/L) 735 837 585 719 127 775 TBIL (μmol/L)2.03 2.71 1.47 2.07 0.62 1.80 DBIL (μmol/L) 0.49 1.00 0.29 0.59 0.370.89 GLU (mmol/L) 4.13 3.17 3.82 3.71 0.49 2.93 GGT (U/L) 119 100 91.1104 14.2 84.1 TP (g/L) 64.5 60.5 67.3 64.1 3.41 62.5 TG (mmol/L) 0.310.19 0.70 0.40 0.27 0.45 BUN (mmol/L) 13.4 11.5 17.2 14.0 2.93 11.5 CREA(μmol/L) 61.0 59.3 79.4 66.6 11.1 64.3 Treatment group G4: ETD01826Animal No. 402 403 Mean SD Gender Male Male Animal ID SC1604087SC1703023 Parameters ALT (U/L) 32.4 12.0 21.3 10.31 (unit) AST (U/L)28.0 28.7 28.3 0.36 ALP (U/L) 346 492 538 218 TBIL (μmol/L) 3.67 1.492.32 1.18 DBIL (μmol/L) 2.40 0.70 1.33 0.93 GLU (mmol/L) 4.14 3.49 3.520.61 GGT (U/L) 69.5 47.2 66.9 18.6 TP (g/L) 61.2 50.3 58.0 6.68 TG(mmol/L) 0.22 0.53 0.40 0.16 BUN (mmol/L) 11.9 17.9 13.7 3.59 CREA(μmol/L) 90.2 85.5 80.0 13.8 Note: The ↓ next to the value means theresult was slightly lower than that of other animals.

TABLE 11 Individual and Mean Clinical Chemistry Parameters Results onPre-dose (Day-2) Treatment group G1: ETD01821 G2: ETD01822 Animal No.101 102 103 201 202 203 Gender Male Male Male Male Male Male Animal IDSC1702037 SC1509029 175151C Mean SD SC1508015 SC1704115 SC1703011 MeanSD Param- ALT 34.4 23.3 45.4 34.4 11.1 21.0 31.5 33.4 28.6 6.68 eters(U/L) (unit) AST 20.9 20.3 44.7 28.6 13.9 20.1 30.0 34.0 28.0 7.16 (U/L)ALP 477 251↓ 634 454 192 279↓ 489 630 466 177 (U/L) TBIL 1.83 1.48 1.071.46 0.38 1.52 2.07 2.69 2.09 0.59 (μmol/ L) DBIL 0.26 0.05 0.02 0.110.13 0.01 0.38 0.58 0.32 0.29 (μmol/ L) GLU 3.31 3.56 3.41 3.43 0.134.40 3.15 3.45 3.67 0.65 (mmol/ L) GGT 104 79.4 97.2 93.6 12.7 98.2 70.268.7 79.0 16.6 (U/L) TP 79.9 74.9 77.0 77.3 2.53 79.3 76.6 77.2 77.71.43 (g/L) TG 0.73 0.47 1.48 0.89 0.52 0.47 0.24 0.47 0.39 0.13 (mmol/L) BUN ) 13.8 11.9 13.9 13.2 1.12 14.1 11.1 15.4 13.5 2.22 (mmol/ L)CREA 85.4 70.8 66.1 74.1 10.1 86.5 63.3 81.4 77.1 12.2 (μmol/ L)Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402403 Gender Male Male Male Male Male Male Animal ID 177695C SC1704077176313C Mean SD SC1708089 SC1604087 SC1703023 Mean SD Param- ALT 32.642.6 38.3 37.8 5.02 24.5 42.5 11.9 26.3 15.4 eters (U/L) (unit) AST 28.730.9 33.0 30.9 2.15 25.7 31.7 24.1 27.2 4.01 (U/L) ALP 679 821 580 693121 762 478 427 556 180 (U/L) TBIL 2.89 2.74 2.33 2.65 0.29 2.15 1.890.74 1.59 0.75 (μmol/ L) DBIL 0.72 0.39 0.69 0.60 0.18 0.51 0.21 — 0.360.21 (μmol/ L) GLU 3.50 3.76 3.85 3.70 0.18 3.98 6.44 4.04 4.82 1.40(mmol/ L) GGT 116 100 96.9 104 10.0 86.4 86.3 50.6 74.4 20.6 (U/L) TP(g/ 70.8 65.4 71.5 69.2 3.31 67.9 75.8 52.7 65.5 11.7 L) TG 0.32 0.240.39 0.32 0.08 0.48 0.32 1.08 0.63 0.40 (mmol/ L) BUN 12.7 10.3 17.513.5 3.66 12.6 11.9 16.0 13.5 2.16 (mmol/ L) CREA 60.1 57.7 74.6 64.19.14 63.0 106 69.8 79.5 22.9 (μmol/ L) Note: The ↓ next to the valuemeans the result was slightly lower than that of other animals. '—'means that DBIL of some samples cannot be detected due to the lowconcentration.

TABLE 12 Individual and Mean Clinical Chemistry Parameters Results onDay 7 post-dose Treatment group G1: ETD01821 G2: ETD01822 Animal No. 101102 103 201 202 203 Gender Male Male Male Male Male Male Animal IDSC1702037 SC1509029 175151C Mean SD SC1508015 SC1704115 SC1703011 MeanSD Param- ALT 32.5 22.7 38.2 31.1 7.84 19.9 36.2 28.7 28.3 8.16 eters(U/L) (unit) AST 22.3 20.0 32.1 24.8 6.43 16.4 34.2 25.4 25.3 8.90 (U/L)ALP 453 276↓ 749 493 239 272↓ 535 628 479 185 (U/L) TBIL 1.56 1.50 1.921.66 0.23 1.66 2.22 2.11 2.00 0.30 (μmol/ L) DBIL 0.09 — 0.30 0.20 0.150.58 — 0.97 0.78 0.28 (μmol/ L) GLU 3.69 3.91 3.83 3.81 0.11 4.49 4.534.32 4.45 0.11 (mmol/ L) GGT 104 78.4 103 95.1 14.4 90.8 71.2 64.5 75.513.7 (U/L) TP 82.8 78.8 82.8 81.4 2.30 76.1 80.8 76.5 77.8 2.58 (g/L) TG0.64 0.59 0.81 0.68 0.12 0.30 0.34 0.65 0.43 0.19 (mmol/L) BUN 14.1 10.513.9 12.8 2.02 11.8 11.2 14.2 12.4 1.58 (mmol/L) CREA 82.1 68.0 62.871.0 9.99 85.0 67.7 76.9 76.5 8.66 (μmol/ L) Treatment group G3:ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402 403 Gender MaleMale Male Male Male Male Animal ID 177695C SC1704077 176313C Mean SDSC1708089 SC1604087 SC1703023 Mean SD Param- ALT 29.9 41.6 30.7 34.16.54 23.0 49.4 15.1 29.2 18.0 eters (U/L) (unit) AST 28.0 29.6 26.2 27.91.70 24.5 28.2 21.4 24.7 3.40 (U/L) ALP 806 776 570 718 129 659 510 433534 115 (U/L) TBIL 2.70 1.91 1.79 2.13 0.49 2.15 2.29 1.61 2.02 0.36(μmol/ L) DBIL 0.39 0.81 0.61 0.60 0.21 0.30 0.31 0.60 0.40 0.17 (μmol/L) GLU 3.93 4.27 3.86 4.02 0.22 3.64 4.60 4.41 4.22 0.51 (mmol/ L) GGT129 96.2 87.0 103.9 21.8 82.0 94.0 55.9 77.3 19.5 (U/L) TP 75.2 66.473.4 71.7 4.67 71.4 83.0 58.6 71.0 12.2 (g/L) TG 0.36 0.37 0.47 0.400.06 0.47 0.27 0.46 0.40 0.11 (mmol/ L) BUN 11.7 10.2 18.7 13.6 4.5213.7 13.6 14.0 13.8 0.19 (mmol/ L) CREA 65.8 55.4 70.7 64.0 7.81 60.9101 75.6 79.0 20.0 (μmol /L) Note: The ↓ next to the value means theresult was slightly lower than that of other animals. '—' means thatDBIL of some samples cannot be detected due to the low concentration.

TABLE 13 Individual and Mean Clinical Chemistry Parameters Results onDay 14 post-dose Treatment group G1: ETD01821 G2: ETD01822 Animal No.101 102 103 201 202 203 Gender Male Male Male Male Male Male Animal IDSC1702037 SC1509029 175151C Mean SD SC1508015 SC1704115 SC1703011 MeanSD Param- ALT 37.0 25.4 66.9 43.1 21.4 20.0 30.4 29.1 26.5 5.67 eters(U/L) (unit) AST 26.0 20.5 178 74.9 89.6 18.2 26.1 25.5 23.3 4.40 (U/L)ALP 557 275↓ 781 538 254 298↓ 509 630 479 168 (U/L) TBIL 1.60 1.74 1.821.72 0.11 1.73 1.88 2.18 1.93 0.23 (μmol/ L) DBIL — 0.28 — 0.28 0.00 —0.40 0.73 0.57 0.23 (μmol/ L) GLU 3.32 3.50 5.43 4.08 1.17 4.57 3.524.01 4.03 0.53 (mmol/ L) GGT 118 76.7 101 98.6 20.8 102 66.3 63.3 77.121.4 (U/L) TP 88.7 76.8 83.9 83.1 5.97 80.2 79.4 75.2 78.3 2.69 (g/L) TG1.09 0.53 0.60 0.74 0.31 0.32 0.24 0.65 0.40 0.22 (mmol/ L) BUN 14.010.1 16.5 13.5 3.25 15.9 11.7 13.3 13.6 2.13 (mmol/ L) CREA 98.9 67.869.1 78.6 17.6 95.2 69.8 78.7 81.2 12.9 (μmol/ L) Treatment group G3:ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402 403 Gender MaleMale Male Male Male Male Animal ID 177695C SCI704077 176313C Mean SDSC1708089 SC1604087 SC1703023 Mean SD Param- ALT 34.6 47.0 23.1 34.912.0 22.1 16.0 46.3 28.1 16.0 eters (U/L) (unit) AST 26.3 30.7 25.0 27.32.99 29.1 29.6 27.9 28.9 0.87 (U/L) ALP 782 864 569 738 153 679 467 469538 122 (U/L) TBIL 1.92 2.66 1.63 2.07 0.53 1.43 0.88 1.78 1.36 0.45(μmol/ L) DBIL 0.37 0.65 0.46 0.49 0.14 0.27 — 0.22 0.25 0.04 (μmol/ L)GLU 3.59 3.70 4.04 3.78 0.23 3.29 4.64 4.18 4.04 0.69 (mmol/ L) GGT 128101 96.4 108 17.1 75.1 54.3 83.7 71.0 15.1 (U/L) TP 71.2 67.8 71.7 70.22.13 65.1 53.6 73.5 64.1 9.99 (g/L) TG 0.38 0.30 0.59 0.42 0.15 0.560.78 0.26↓ 0.53 0.26 (mmol/ L) BUN 12.3 10.3 15.9 12.8 2.82 13.1 18.113.7 15.0 2.78 (mmol/ L) CREA 66.1 61.7 74.8 67.5 6.67 65.1 92.2 10085.8 18.3 (μmol/ L) Note: The ↓ next to the value means the result wasslightly lower than that of other animals. '—' means that DBIL of somesamples cannot be detected due to the low concentration.

TABLE 14 Individual and Mean Clinical Chemistry Parameters Results onDay 28 post-dose Treatment group G1: ETD01821 G2: ETD01822 Animal No.101 102 103 201 202 203 Gender Male Male Male Male Male Male Animal IDSC1702037 SC1509029 175151C Mean SD SC1508015 SC1704115 SC1703011 MeanSD Param- ALT 34.8 22.1 45.3 34.1 11.6 33.5 45.4 48.8 42.6 8.03 eters(U/L) (unit) AST 30.1 30.4 35.0 31.8 2.75 25.8 42.4 51.9 40.0 13.2 (U/L)ALP 364 216↓ 503 361 144 226↓ 467 634 442 205 (U/L) TBIL 1.17 1.33 1.151.22 0.10 1.72 1.08 1.09 1.30 0.37 (μmol/ L) DBIL 0.10 0.33 0.28 0.240.12 0.29 — — 0.29 0.00 (μmol/ L) GLU 3.06 2.97 3.04 3.02 0.05 4.56 3.794.61 4.32 0.46 (mmol/ L) GGT 87.8 66.5 79.8 78.0 10.7 86.6 63.9 62.370.9 13.6 (U/L) TP 72.3 72.4 71.7 72.1 0.36 82.4 74.7 72.4 76.5 5.23(g/L) TG 0.79 0.43 0.62 0.61 0.18 0.41 0.13 0.40 0.31 0.16 (mmol/ L) BUN18.2 14.1 15.9 16.1 2.05 16.1 11.4 15.5 14.3 2.54 (mmol/ L) CREA 75.063.0 65.9 68.0 6.26 89.3 60.3 76.9 75.5 14.6 (μmol/ L) Treatment groupG3: ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402 403 Gender MaleMale Male Male Male Male Animal ID 177695C SC1704077 176313C Mean SDSC1708089 SC1604087 SC1703023 Mean SD Param- ALT 23.9 34.3 28.4 28.95.22 22.8 40.5 24.4 29.2 9.79 eters (U/L) (unit) AST 24.5 43.1 27.1 31.610.1 31.5 30.3 34.6 32.1 2.22 (U/L) ALP 193 447 563 401 189 570 344 382432 121 (U/L) TBIL 1.53 1.69 1.30 1.51 0.20 1.37 0.96 0.58 0.97 0.40(μmol/ L) DBIL 0.45 0.37 0.56 0.46 0.10 — 0.05 — 0.05 0.00 (μmol/ L) GLU3.83 3.21 3.30 3.45 0.34 2.97 5.57 4.58 4.37 1.31 (mmol/ L) GGT 77.265.3 59.2 67.2 9.13 73.5 75.9 53.8 67.7 12.1 (U/L) TP 73.5 73.7 68.171.7 3.17 65.0 62.4 58.1 61.8 3.48 (g/L) TG 0.28 0.17 0.42 0.29 0.130.54 0.15 0.36 0.35 0.20 (mmol/ L) BUN 16.0 11.2 14.9 14.0 2.53 14.816.9 16.2 16.0 1.05 (mmol/ L) CREA 86.3 55.1 74.3 71.9 15.7 62.4 96.862.2 73.8 19.9 (μmol/ L) Note: The ↓ next to the value means the resultwas slightly lower than that of other animals. '—' means that DBIL ofsome samples cannot be detected due to the low concentration.

TABLE 15 Individual and Mean Hematology Results on Pre-dose (Day-8)Treatment group G1: ETD01821 G2: ETD01822 Animal No. 101 102 103 201 202203 Gender Male Male Male Male Male Male Animal ID SC1702037 SC1509029175151C Mean SD SC1508015 SC1704115 SC1703011 Mean SD Param- WBC 10.47.99 8.57 8.99 1.26 6.69 10.4 17.4 11.5 5.43 eters (×10⁹/L) (unit) abs_3.14 2.59 1.33 2.35 0.93 2.52 2.97 3.13 2.87 0.32 neuts (×10⁹/L) abs_6.55 4.82 6.61 5.99 1.02 3.76 6.77 13.48 8.00 4.98 lymphs (×10⁹/L) abs_0.65 0.48 0.46 0.53 0.10 0.31 0.59 0.66 0.52 0.19 monos (×10⁹/L) abs_eos0.07 0.10 0.17 0.11 0.05 0.10 0.04 0.10 0.08 0.03 (×10⁹/L) abs_basos0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (×10⁹/L) % NEUT 30.132.4 15.6 26.0 9.11 37.6 28.6 18.0 28.1 9.81 (%) % LYM 63.0 60.4 77.066.8 8.93 56.2 65.3 77.6 66.4 10.7 (%) % MONO 6.20 6.00 5.40 5.87 0.424.70 5.70 3.80 4.73 0.95 (%) % EOS 0.70 1.20 2.00 1.30 0.66 1.50 0.400.60 0.83 0.59 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 (%) RBC 5.43 5.48 5.35 5.42 0.07 5.01 5.33 5.29 5.21 0.17 (×10¹²/L)HGB 135 134 124 131 6.08 126 128 131 128 2.52 (g/L) HCT 45.2 43.5 40.443.0 2.43 41.0 42.1 42.5 41.9 0.78 (%) MCV 83.3 79.5 75.6 79.5 3.85 82.079.1 80.4 80.5 1.45 (fL) MCH 24.8 24.4 23.2 24.1 0.83 25.2 24.1 24.924.7 0.57 (pg) MCHC 298 307 307 304 5.20 308 304 309 307 2.65 (g/L) RDW-37.1 43.6 38.8 39.8 3.37 41.2 36.7 36.9 38.3 2.54 SD (fL) RDW- 12.2 15.114.1 13.8 1.47 13.7 12.7 12.6 13.0 0.61 CV (%) PLT 380 371 247↓ 333 74.3361 301 285 316 40.1 (×10⁹/L) MPV (fL) 13.1 12.5 9.60 11.7 1.87 11.614.2 12.4 12.7 1.33 PCT (%) 0.50 0.46 0.24↓ 0.40 0.14 0.42 0.43 0.360.40 0.04 PDW fL) 15.5 15.0 15.4 15.3 0.26 14.9 15.6 15.3 15.3 0.35Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402403 Gender Male Male Male Male Male Male Animal ID 177695C SC1704077176313C Mean SD SC1708089 SC1604087 SC1703023 Mean SD Param- WBC 13.99.37 8.40 10.6 2.93 9.50 5.65↓ 15.8 10.3 5.13 eters (×10⁹/L) (unit) abs_1.19 2.31 1.82 1.77 0.56 3.72 1.04↓ 6.22 3.66 2.59 neuts (×10⁹/L) abs_11.3 6.45 6.09 7.96 2.93 5.09 4.13 8.76 5.99 2.44 lymphs (×10⁹/L) abs_1.28 0.53 0.29 0.70 0.52 0.55 0.38 0.57 0.50 0.10 monos (×10⁹/L) abs_eos0.09 0.08 0.20 0.12 0.07 0.14 0.10 0.26 0.17 0.08 (×10⁹/L) abs_basos0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.01 (×10⁹/L) % NEUT 8.624.7 21.6 18.3 8.54 39.2 18.5 39.3 32.3 12.0 (%) % LYM 81.5 68.9 72.674.3 6.48 53.5 72.9 55.4 60.6 10.7 (%) % MONO 9.20 5.60 3.40 6.07 2.935.80 6.80 3.60 5.40 1.64 (%) % EOS 0.70 0.80 2.40 1.30 0.95 1.50 1.801.70 1.67 0.15 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 (%) RBC 5.36 5.61 5.07 5.35 0.27 5.29 5.09 5.39 5.26 0.15 (×10¹²/L)HGB 130 137 114 127 11.8 129 122 133 128 5.57 (g/L) HCT 43.6 43.3 37.341.4 3.55 42.2 40.3 43.3 41.9 1.52 (%) MCV 81.3 77.3 73.6 77.4 3.85 79.679.2 80.3 79.7 0.56 (fL) MCH 24.2 24.5 22.6 23.8 1.02 24.3 24.1 24.624.3 0.25 (pg) MCHC 298 316 307 307 9.00 305 304 306 305 1.00 (g/L) RDW-41.1 38.9 36.5 38.8 2.30 36.9 40.2 40.3 39.1 1.93 SD (fL) RDW- 13.8 13.813.6 13.7 0.12 12.7 14.0 13.8 13.5 0.70 CV (%) PLT 372 307 229↓ 303 71.6339 201↓ 398 313 101 (×10⁹/L) MPV (fL) 11.1 11.9 14.4 12.5 1.72 13.114.2 10.3 12.5 2.01 PCT (%) 0.41 0.37 0.33 0.37 0.04 0.44 0.29↓ 0.410.38 0.08 PDW (fL) 15.5 15.3 15.8 15.5 0.25 15.5 15.6 15.3 15.5 0.15Note: The ↓ next to the value means the result was slightly lower thanthat of other animals.

TABLE 16 Individual and Mean Hematology Results on Pre-dose (Day-2)Treatment group G1: ETD01821 G2: ETD01822 Animal No. 101 102 103 201 202203 Gender Male Male Male Male Male Male Animal ID SC1702037 SC1509029175151C Mean SD SC1508015 SC1704115 SC1703011 Mean SD Param- WBC 8.9814.8 15.4 13.1 3.55 7.48 13.5 19.3 13.4 5.90 eters (×10⁹/L) (unit)abs_neuts 3.44 6.07 5.05 4.85 1.33 3.20 4.94 4.64 4.26 0.93 (×10⁹/L)abs_lymphs 4.69 7.74 9.41 7.28 2.39 3.80 7.46 13.62 8.29 4.96 (×10⁹/L)abs_monos 0.76 0.76 0.75 0.76 0.01 0.41 0.99 0.96 0.79 0.33 (×10⁹/L)abs_eos 0.09 0.20 0.22 0.17 0.07 0.07 0.08 0.05 0.07 0.02 (×10⁹/L)abs_basos 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 (×10⁹/L) %NEUT 38.3 41.1 32.7 37.4 4.28 42.8 36.7 24.1 34.5 9.54 (%) % LYM 52.352.4 61.1 55.3 5.05 50.9 55.3 70.6 58.9 10.3 (%) % MONO 8.40 5.20 4.806.13 1.97 5.40 7.40 5.00 5.93 1.29 (%) % EOS 1.00 1.30 1.40 1.23 0.210.90 0.60 0.30 0.60 0.30 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 (%) RBC 5.63 5.70 5.95 5.76 0.17 5.68 5.75 5.54 5.66 0.11(×10¹²/L) HGB 142 144 140 142 2.00 145 143 141 143 2.00 (g/L) HCT 47.146.1 45.4 46.2 0.85 47.2 45.9 44.9 46.0 1.15 (%) MCV 83.7 80.8 76.3 80.33.73 83.1 79.8 81.1 81.3 1.66 (fL) MCH 25.2 25.2 23.5 24.6 0.98 25.524.8 25.4 25.2 0.38 (pg) MCHC 301 312 308 307 5.57 307 311 313 310 3.06(g/L) RDW-SD 36.7 43.3 38.8 39.6 3.37 41.1 37.1 36.9 38.4 2.37 (fL) RDW-12.1 14.7 13.9 13.6 1.33 13.6 12.7 12.6 13.0 0.55 CV (%) PLT 468 258 292339 113 346 354 343 348 5.7 (×10⁹/L) MPV 11.0 13.5 11.0 11.8 1.44 12.013.1 11.5 12.2 0.82 (fL) PCT 0.51 0.35 0.32 0.39 0.10 0.42 0.47 0.400.43 0.04 (%) PDW 15.1 15.6 15.4 15.4 0.25 15.6 15.7 15.4 15.6 0.15 (fL)Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402403 Gender Male Male Male Male Male Male Animal ID 177695C SC1704077176313C Mean SD SC1708089 SC1604087 SC1703023 Mean SD Param- WBC 10.912.0 12.1 11.7 0.70 11.4 9.31 19.0 13.2 5.13 eters (×10⁹/L) (unit)abs_neuts 1.34 2.94 3.64 2.64 1.18 3.68 1.59 7.33 4.20 2.91 (×10⁹/L)abs_ 8.59 8.05 7.86 8.17 0.38 6.76 6.87 10.76 8.13 2.28 lymphs (×10⁹/L)abs_ 0.86 0.87 0.38 0.70 0.28 0.66 0.70 0.51 0.62 0.10 monos (×10⁹/L)abs_eos 0.06 0.12 0.25 0.14 0.10 0.27 0.14 0.44 0.28 0.15 (×10⁹/L)abs_basos 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01 (×10⁹/L) %NEUT 12.3 24.6 30.0 22.3 9.07 32.4 17.1 38.5 29.3 11.0 (%) % LYM 79.267.2 64.8 70.4 7.71 59.4 73.8 56.5 63.2 9.27 (%) % MONO 7.90 7.20 3.206.10 2.54 5.80 7.50 2.70 5.33 2.43 (%) % EOS 0.60 1.00 2.00 1.20 0.722.40 1.50 2.30 2.07 0.49 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.100.00 0.03 0.06 (%) RBC 5.41 5.86 5.34 5.54 0.28 5.69 5.76 5.19 5.55 0.31(×10¹²/L) HGB 132 144 122 133 11.0 140 141 126 136 8.39 (g/L) HCT 44.145.4 39.6 43.0 3.04 45.5 46.0 41.6 44.4 2.41 (%) MCV 81.5 77.5 74.2 77.73.66 80.0 79.9 80.1 80.0 0.10 (fL) MCH 24.5 24.5 22.9 24.0 0.92 24.624.5 24.2 24.4 0.21 (pg) MCHC 300 317 309 309 8.50 308 306 302 305 3.06(g/L) RDW- 41.5 39.1 37.7 39.4 1.92 36.7 40.2 38.9 38.6 1.77 SD (fL)RDW- 14.0 13.9 13.9 13.9 0.06 12.6 13.9 13.4 13.3 0.66 CV (%) PLT 379349 237↓ 322 74.8 445 302 393 380 72.4 (×10⁹/L) MPV 11.4 11.6 15.4 12.82.25 11.9 15.5 11.0 12.8 2.38 (fL) PCT 0.43 0.41 0.37 0.40 0.03 0.530.47 0.43 0.48 0.05 (%) PDW 16.0 15.1 15.9 15.7 0.49 15.7 15.5 15.9 15.70.20 (fL) Note: The ↓ next to the value means the result was slightlylower than that of other animals.

TABLE 17 Individual and Mean Hematology Results on Day 7 post-doseTreatment group G1: ETD01821 G2: ETD01822 Animal No. 101 102 103 201 202203 Gender Male Male Male Male Male Male Animal ID SC1702037 SC1509029175151C Mean SD SC1508015 SC1704115 SC1703011 Mean SD Param- WBC 8.5213.8 11.5 11.3 2.67 8.62 12.3 17.4 12.8 4.43 eters (×10⁹/L) (unit)abs_neuts 2.00 4.12 2.54 2.89 1.10 4.27 3.44 3.85 3.85 0.42 (×10⁹/L)abs_ 5.75 8.63 8.17 7.52 1.55 3.88 8.07 12.64 8.20 4.38 lymphs (×10⁹/L)abs_ 0.64 0.70 0.61 0.65 0.05 0.43 0.68 0.87 0.66 0.22 monos (×10⁹/L)abs_eos 0.13 0.39 0.21 0.24 0.13 0.04 0.06 0.08 0.06 0.02 (×10⁹/L)abs_basos 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01 (×10⁹/L) %NEUT 23.4 29.7 22.1 25.1 4.06 49.5 28.1 22.1 33.2 14.4 (%) % LYM 67.662.4 70.8 66.9 4.24 45.1 65.8 72.4 61.1 14.2 (%) % MONO 7.50 5.10 5.305.97 1.33 5.00 5.50 5.00 5.17 0.29 (%) % EOS 1.50 2.80 1.80 2.03 0.680.40 0.50 0.50 0.47 0.06 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.100.00 0.03 0.06 (%) RBC ) 5.49 5.70 6.24 5.81 0.39 5.75 6.04 5.54 5.780.25 (×10¹²/L HGB 141 146 146 144 2.89 146 149 140 145 4.58 (g/L) HCT46.2 46.5 47.7 46.8 0.79 47.2 49.0 45.0 47.1 2.00 (%) MCV 84.0 81.5 76.480.6 3.87 82.2 81.1 81.3 81.5 0.59 (fL) MCH 25.6 25.7 23.5 24.9 1.2425.4 24.6 25.3 25.1 0.44 (pg) MCHC 305 315 307 309 5.29 309 303 311 3084.16 (g/L) RDW- 37.8 44.4 39.3 40.5 3.46 40.4 38.4 38.3 39.0 1.18 SD(fL) RDW- 12.3 15.0 14.1 13.8 1.37 13.5 13.1 13.0 13.2 0.26 CV (%) PLT527 390 253↓ 390 137 371 283 341 332 44.7 (×10⁹/L) MPV 12.6 13.7 11.812.7 0.95 12.1 15.3 11.7 13.0 1.97 (fL) PCT 0.67 0.54 0.30 0.50 0.190.45 0.43 0.40 0.43 0.02 (%) PDW 15.4 15.3 15.8 15.5 0.26 15.3 15.4 15.415.4 0.06 (fL) Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301302 303 401 402 403 Gender Male Male Male Male Male Male Animal ID177695C SC1704077 176313C Mean SD SC1708089 SC1604087 SC1703023 Mean SDParam- WBC 9.86 14.2 9.42 11.2 2.63 10.8 8.50 16.7 12.0 4.22 eters(×10⁹/L) (unit) abs_neuts 1.06 3.85 2.05 2.32 1.41 4.77 1.67 5.55 4.002.05 (×10⁹/L) abs_ 7.99 9.18 6.90 8.02 1.14 5.25 6.17 10.3 7.23 2.68lymphs (×10⁹/L) abs_monos 0.73 1.04 0.29 0.69 0.38 0.61 0.52 0.51 0.550.06 (×10⁹/L) abs_eos 0.08 0.11 0.18 0.12 0.05 0.21 0.14 0.35 0.23 0.11(×10⁹/L) abs_basos 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00(×10⁹/L) % NEUT 10.8 27.1 21.7 19.9 8.30 44.0 19.6 33.3 32.3 12.2 (%) %LYM 81.0 64.8 73.3 73.0 8.10 48.4 72.7 61.6 60.9 12.2 (%) % MONO 7.407.30 3.10 5.93 2.45 5.60 6.10 3.00 4.90 1.66 (%) % EOS 0.80 0.80 1.901.17 0.64 2.00 1.60 2.10 1.90 0.26 (%) % BASO 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 (%) RBC 5.74 5.42 5.58 5.58 0.16 5.59 6.15 5.625.79 0.32 (×10¹²/L) HGB 143 133 130 135 6.81 141 151 136 143 7.64 (g/L)HCT 46.9 42.1 41.0 43.3 3.14 45.0 49.2 45.0 46.4 2.42 (%) MCV 81.8 77.773.5 77.7 4.15 80.6 80.1 80.0 80.2 0.32 (fL) MCH 24.9 24.5 23.3 24.20.83 25.3 24.6 24.2 24.7 0.56 (pg) MCHC 304 315 317 312 7.00 314 307 303308 5.57 (g/L) RDW- 42.9 39.3 36.4 39.5 3.26 37.9 41.3 39.3 39.5 1.71 SD(fL) RDW- 14.4 13.9 13.6 14.0 0.40 12.9 14.2 13.5 13.5 0.65 CV (%) PLT441 317 256↓ 338 94.3 478 258↓ 369 368 110 (×10⁹/L) MPV 10.7 12.5 15.012.7 2.16 12.2 15.6 11.5 13.1 2.19 (fL) PCT 0.47 0.40 0.39 0.42 0.050.59 0.40 0.42 0.47 0.10 (%) PDW 15.6 15.3 15.9 15.6 0.30 15.3 15.3 15.815.5 0.29 (fL) Note: The ↓ next to the value means the result wasslightly lower than that of other animals.

TABLE 18 Individual and Mean Hematology Results on Day 14 post-doseTreatment group G1: ETD01821 G2: ETD01822 Animal No. 101 102 103 201 202203 Gender Male Male Male Male Male Male Animal ID SC1702037 SC150902975151C Mean SD SC1508015 SC1704115 SC1703011 Mean SD Param- WBC 9.9310.4 20.7 13.7 6.08 12.0 12.1 17.9 14.0 3.39 eters (×10⁹/L) (unit)abs_neuts 1.82 3.09 13.5↑ 6.13 6.40 7.15 2.75 3.78 4.56 2.30 (×10⁹/L)abs_ 7.15 6.21 5.59 6.32 0.79 4.26 8.44 12.97 8.56 4.36 lymphs (×10⁹/L)abs_ 0.81 0.73 1.53 1.02 0.44 0.55 0.86 1.06 0.82 0.26 monos (×10⁹/L)abs_eos 0.14 0.35 0.07 0.19 0.15 0.04 0.05 0.11 0.07 0.04 (×10⁹/L)abs_basos 0.01 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.01 (×10⁹/L) %NEUT 18.3 29.8 65.2↑ 37.8 24.4 59.6 22.7 21.1 34.5 21.8 (%) % LYM 72.159.7 27.1 53.0 23.2 35.5 69.8 72.4 59.2 20.6 (%) % MONO 8.10 7.10 7.407.53 0.51 4.60 7.10 5.90 5.87 1.25 (%) % EOS 1.40 3.40 0.30 1.70 1.570.30 0.40 0.60 0.43 0.15 (%) % BASO 0.10 0.00 0.00 0.03 0.06 0.00 0.000.00 0.00 0.00 (%) RBC 5.99 5.33 6.52 5.95 0.60 5.84 6.02 5.39 5.75 0.32(×10¹²/L) HGB 152 140 157 150 8.74 151 151 138 147 7.51 (g/L) HCT 50.144.1 49.3 47.8 3.26 49.3 48.5 44.3 47.4 2.69 (%) MCV 83.7 82.7 75.7 80.74.36 84.5 80.6 82.2 82.4 1.96 (fL) MCH 25.4 26.3 24.1 25.3 1.11 25.925.0 25.7 25.5 0.47 (pg) MCHC 304 318 318 313 8.08 307 311 312 310 2.65(g/L) RDW-SD 37.0 44.1 38.1 39.7 3.82 41.5 37.8 39.1 39.5 1.88 (IL)RDW-CV 12.2 14.6 13.9 13.6 1.23 13.5 12.9 13.2 13.2 0.30 (%) PLT 570 311192↓ 358 193 279 240 322 280 41.0 (x10⁹/L) MPV 12.3 13.8 11.80 12.6 1.0412.8 15.0 11.80 13.2 1.64 (fL) PCT 0.70 0.43 0.23↓ 0.45 0.24 0.36 0.360.38 0.37 0.01 (%) PDW 14.9 15.5 15.9 15.4 0.50 15.8 15.7 15.6 15.7 0.10(fL) Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301 302 303401 402 403 Gender Male Male Male Male Male Male Animal ID 177695CSC1704077 176313C Mean SD SC1708089 SC1604087 SC1703023 Mean SD Param-WBC 9.24 12.1 10.0 10.4 1.45 10.3 7.73 16.9 11.7 4.74 eters (×10⁹/L)(unit) abs_neuts 0.54↓ 2.62 3.13 2.10 1.37 4.47 1.34 5.81 3.87 2.29(×10⁹/L) abs_ 7.95 8.52 6.34 7.60 1.13 5.08 5.65 10.25 6.99 2.83 lymphs(×10⁹/L) abs_monos 0.67 0.85 0.30 0.61 0.28 0.59 0.57 0.46 0.54 0.07(×10⁹/L) abs_eos 0.08 0.07 0.26 0.14 0.11 0.19 0.17 0.39 0.25 0.12(×10⁹/L) abs_basos 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.01(×10⁹/L) % NEUT 5.90↓ 21.7 31.3 19.6 12.8 43.3 17.4 34.4 31.7 13.2 (%) %LYM 86.0 70.6 63.1 73.2 11.7 49.2 73.0 60.6 60.9 11.9 (%) % MONO 7.307.10 3.00 5.80 2.43 5.70 7.40 2.70 5.27 2.38 (%) % EOS 0.80 0.60 2.601.33 1.10 1.80 2.20 2.30 2.10 0.26 (%) % BASO 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 (%) RBC 5.59 5.58 5.52 5.56 0.04 5.25 5.59 5.685.51 0.23 (×10¹²/L) HGB 141 138 128 136 6.81 131 139 141 137 5.29 (g/L)HCT 45.7 43.9 40.7 43.4 2.53 42.4 45.1 45.5 44.3 1.69 (%) MCV 81.7 78.873.8 78.1 4.00 80.8 80.7 80.1 80.5 0.38 (fL) MCH 25.2 24.8 23.1 24.41.12 24.9 25.0 24.8 24.9 0.10 (pg) MCHC 308 314 313 312 3.21 309 309 310309 0.58 (g/L) RDW- 42.6 40.6 36.6 39.9 3.06 37.1 41.6 38.4 39.0 2.32 SD(fL) RDW- 14.2 14.3 13.6 14.0 0.38 12.6 14.2 13.2 13.3 0.81 CV (%) PLT376 278 218↓ 291 79.8 380 250↓ 460 363 106 (×10⁹/L) MPV 10.2 12.6 16.113.0 2.97 11.9 15.1 10.2 12.4 2.49 (fL) PCT 0.38 0.35 0.35 0.36 0.020.45 0.38 0.47 0.43 0.05 (%) PDW 15.5 15.5 15.7 15.6 0.12 15.7 15.5 15.515.6 0.12 (fL) Note: The ↓ next to the value means the result wasslightly lower than that of other animals.

TABLE 19 Individual and Mean Hematology Results on Day 28 post-doseTreatment group G1: ETD01821 G2: ETD01822 Animal No. 101 102 103 201 202203 Gender Male Male Male Male Male Male Animal ID SC1702037 SC1509029175151C Mean SD SC1508015 SC1704115 SC1703011 Mean SD Pa- WBC 5.30↓ 8.449.93 7.89 2.36 12.4 12.6 11.7 12.2 0.47 ram- (×10⁹/L) eters abs_ 1.753.52 4.22 3.16 1.27 8.91 7.55 4.53 7.00 2.24 (unit) neuts ) (×10⁹/L abs_3.20 4.41 5.27 4.29 1.04 2.78 4.40 6.49 4.56 1.86 lymphs (×10⁹/L) abs_0.32 0.39 0.28 0.33 0.06 0.67 0.62 0.66 0.65 0.03 monos (×10⁹/L) abs_eos0.03 0.12 0.16 0.10 0.07 0.02 0.02 0.01 0.02 0.01 (×10⁹/L) abs_ 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 basos (×10⁹/L) % NEUT 33.141.7 42.5 39.1 5.21 72.0 59.9 38.8 56.9 16.8 (%) % LYM 60.3 52.3 53.155.2 4.41 22.5 34.9 55.4 37.6 16.6 (%) % 6.00 4.60 2.80 4.47 1.60 5.405.00 5.70 5.37 0.35 MONO (%) % EOS 0.60 1.40 1.60 1.20 0.53 0.10 0.200.10 0.13 0.06 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 (%) RBC 5.15 5.19 5.09 5.14 0.05 5.90 5.61 5.46 5.66 0.22 (×10¹²/L)HGB 131 136 121 129 7.64 154 140 142 145 7.57 (g/L) HCT 42.6 43.6 38.541.6 2.70 49.6 44.8 44.7 46.4 2.80 (%) MCV 82.8 84.0 75.7 80.8 4.49 84.179.8 82.0 82.0 2.15 (fL) MCH 25.4 26.2 23.7 25.1 1.28 26.1 25.0 26.125.7 0.64 (pg) MCHC 307 312 313 311 3.21 311 313 318 314 3.61 (g/L) RDW-36.4 41.3 37.2 38.3 2.63 41.0 37.4 39.7 39.4 1.82 SD (fL) RDW- 12.1 13.513.5 13.0 0.81 13.4 12.9 13.3 13.2 0.26 CV (%) PLT 480 368 329 392 78.4399 355 346 367 28.4 (×10⁹/L) MPV 11.2 14.2 10.4 11.9 2.00 11.6 14.011.5 12.4 1.42 (fL) PCT 0.54 0.53 0.34 0.47 0.11 0.46 0.50 0.40 0.450.05 (%) PDW 14.9 15.3 15.2 15.1 0.21 15.5 15.6 15.4 15.5 0.10 (fL)Treatment group G3: ETD01823 G4: ETD01826 Animal No. 301 302 303 401 402403 Gender Male Male Male Male Male Male Animal ID 177695C SC170407776313C Mean SD SC1708089 SC1604087 SC1703023 Mean SD Pa- WBC 6.49↓ 12.514.2 11.1 4.05 6.51↓ 7.22 12.7 8.79 3.36 ram- (×10⁹/L) eters abs_ 3.844.42 4.13 4.13 0.29 3.62 4.15 6.51 4.76 1.54 (unit) neuts (×10⁹/L) abs_2.27↓ 7.35 9.37 6.33 3.66 2.53↓ 2.62↓ 5.64 3.60 1.77 lymphs (×10⁹/L)abs_ 0.36 0.65 0.69 0.57 0.18 0.30 0.37 0.34 0.34 0.04 monos (×10⁹/L)abs_eos 0.02 0.05 0.03 0.03 0.02 0.06 0.08 0.15 0.10 0.05 (×10⁹/L) abs_0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.01 basos (×10⁹/L) % NEUT59.2 35.5 29.0 41.2 15.9 55.6 57.5 51.5 54.9 3.07 (%) % LYM 35.0 58.965.9 53.3 16.2 38.9 36.2 44.5 39.9 4.23 (%) % 5.50 5.20 4.90 5.20 0.304.60 5.10 2.70 4.13 1.27 MONO (%) % EOS 0.30 0.40 0.20 0.30 0.10 0.901.20 1.20 1.10 0.17 (%) % BASO 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.100.03 0.06 (%) RBC 5.02 5.78 4.91 5.24 0.47 5.12 5.05 5.07 5.08 0.04(×10¹²/L) HGB 132 143 126 134 8.62 128 126 124 126 2.00 (g/L) HCT 42.145.2 40.4 42.6 2.43 41.1 40.5 40.6 40.7 0.32 (%) MCV 83.8 78.2 82.4 81.52.91 80.3 80.2 80.0 80.2 0.15 (fL) MCH 26.3 24.8 25.6 25.6 0.75 25.125.0 24.5 24.9 0.32 (pg) MCHC 314 316 311 314 2.52 312 312 306 310 3.46(g/L) RDW- 40.5 39.1 39.9 39.8 0.70 36.0 41.3 39.5 38.9 2.70 SD (fL)RDW- 13.3 13.8 13.4 13.5 0.26 12.2 14.1 13.6 13.3 0.98 CV (%) PLT 355384 307 349 38.9 402 162↓ 399 321 137.7 (×10⁹/L) MPV 12.4 11.8 11.9 12.00.32 11.8 15.3 9.40 12.2 2.97 (fL) PCT 0.44 0.45 0.37 0.42 0.05 0.480.25 0.38 0.37 0.11 (%) PDW 15.4 15.1 15.2 15.2 0.15 15.5 15.8 15.5 15.60.17 (fL) Note: The ↓ next to the value means the result was slightlylower than that of other animals.

TABLE 20 Relative Mean Serum MSP Level in Cynomolgus Monkeys Day Dose −8−2 7 14 28 42 56 70 77 84 91 98 105 Group n Treatment (mg/kg) Mean SerumMSP Level (Relative to mean of pre-dose level (Day −2 and Day −8)) 1 3ETD01821 5 1.09 0.91 0.22 0.06 0.04 0.07 0.15 0.18 0.18 0.31 0.42 0.450.46 2 3 ETD01822 5 0.99 1.01 0.71 0.32 0.42 0.37 0.76 1.24 1.16 1.481.34 1.34 1.57 3 3 ETD01823 5 1.01 0.99 0.44 0.08 0.34 0.10 0.20 0.450.39 0.52 0.59 0.91 1.03 4 3 ETD01826 5 0.78 1.22 0.37 0.20 0.30 0.580.88 1.01 1.63 1.32 1.86 1.98 1.95

TABLE 21 Relative Individual Serum MSP Level in Cynomolgus Monkeys DayDose Animal −8 −2 7 14 28 42 56 70 77 84 91 98 105 Group n Treatment(mg/kg) # Mean Serum MSP Level (Relative to mean of pre-dose level (Day−2 and Day −8)) 1 3 ETD01821 5 101M 0.99 1.01 0.28 0.08 0.07 0.11 0.23102M 1.12 0.88 0.18 0.07 0.04 0.01 0.16 0.26 0.21 0.31 0.47 0.51 0.39103M 1.16 0.84 0.19 0.02 0.01 0.08 0.07 0.09 0.15 0.31 0.37 0.38 0.52 23 ETD01822 5 201M 0.99 1.01 0.64 0.43 0.75 0.59 0.90 1.26 0.95 1.56 1.691.75 1.49 202M 0.99 1.01 0.75 0.30 0.23 0.29 0.86 1.39 1.58 0.96 1.231.14 1.82 203M 1.00 1.00 0.74 0.24 0.28 0.23 0.53 1.07 0.96 1.92 1.101.13 1.39 3 3 ETD01823 5 301M 0.76 1.24 0.24 0.05 0.50 0.06 0.15 0.190.18 0.36 0.30 0.41 0.56 302M 1.18 0.82 0.46 0.09 0.31 0.15 0.26 0.710.57 0.84 0.83 1.44 1.40 303M 1.09 0.91 0.61 0.11 0.22 0.10 0.20 0.450.42 0.37 0.65 0.89 1.14 4 3 ETD01826 5 401M 0.95 1.05 0.26 0.16 0.250.43 0.91 0.91 1.32 1.51 1.51 2.25 2.16 402M 0.91 1.09 0.35 0.07 0.150.15 0.41 0.77 1.55 0.89 1.23 1.81 1.54 403M 0.49 1.51 0.51 0.36 0.491.16 1.31 1.35 2.02 1.56 2.84 1.87 2.15

TABLE 22 Relative MST1 mRNA Level in Liver of Cynomolgus Monkeys MeanMST1 mRNA Dose (Relative to Day −8) Group n Treatment (mg/kg) Day −8 Day28 1 3 ETD01821 5 1.00 0.33 2 3 ETD01822 5 1.00 0.33 3 3 ETD01823 5 1.000.27 4 3 ETD01826 5 1.00 0.57

Example 19: Screening siRNAs Targeting Human and Mouse MTRES1 in Mice

The following example demonstrates the usefulness of GalNAc moietiesdescribed herein in vivo when combined oligonucleotides targeting anadditional target (MTRES1). Several siRNAs designed to be cross-reactivewith human, mouse and cynomolgus monkey MTRES1 mRNA were tested foractivity in mice. The siRNAs were attached to the GalNAc ligand ETL1 orETL17. The siRNA sequences are shown in Table 23, where Nf is a 2′fluoro-modified nucleoside, n is a 2′ O-methyl modified nucleoside, “d”is a deoxynucleoside, and “s” is a phosphorothioate linkage.

Six to eight week old female mice (strain ICR, n=3) were given asubcutaneous injection on Day 0 of a single 200 ug dose of aGalNAc-conjugated siRNA or PBS as vehicle control.

Mice were euthanized on Day 10 after injection and a liver sample fromeach was collected and placed in RNAlater (ThermoFisher Catalog #AM7020)until processing. Total liver RNA was prepared by homogenizing the livertissue in homogenization buffer (Maxwell RSC simplyRNA Tissue Kit) usinga Percellys 24 tissue homogenizer (Bertin Instruments) set at 5000 rpmfor two 10 second cycles. Total RNA from the lysate was purified on aMaxwell RSC 48 platform (Promega Corporation) according to themanufacturer's recommendations. Preparation of cDNA was performed usingQuanta qScript cDNA SuperMix (VWR, Catalog #95048-500) according to themanufacturer's instructions. The relative levels of liver MIRES1 mRNAwere assessed by RT-qPCR in triplicate on a QuantStudio™ 6 Pro Real-TimePCR System using TaqMan assays for mouse MTRES1 (ThermoFisher, assay#Mm01229834_m1) and the mouse housekeeping gene PPIA (ThermoFisher,assay #Mm02342430_g1) and PerfeCTa® qPCR FastMix), Low ROX™ (VWR,Catalog #101419-222). Data were normalized to the mean MTRES1 mRNA levelin animals receiving PBS. Results are shown in Table 24. Mice injectedwith ETD01597, ETD01955, ETD01958, and had substantially lower levels inmean liver MTRES1 mRNA on Day 10 relative to mice receiving PBS.

TABLE 23 Example siRNA Sequences siRNA SEQ Sense Strand Sequence SEQName ID NO: (5′-3′) with GalNAc moiety ID NO:Antisense Strand Sequence (5′-3′) ETD01597 23 [ETL1]sguaucuccAfgAfauguua33 usAfsuAfaCfaUfuCfuGfgAfgAfuAfcsusu uasusu ETD01954 24[ETL17]sacuuccuGfGfAfAfucg 34 usGfsuAfsuCfgAfuUfcCfaGfgAfaGfususuauacasusu ETD01955 25 [ETL17]scuuccuGfGfAfAfucga 35usAfsgUfaUfcGfaUfuCfcAfgGfaAfgsusu uacuasusu ETD01956 26[ETL17]scuggAfAfucGfAfuacu 36 usUfsaCfaAfgUfaUfcGfaUfuCfcAfgsusuuguaasusu ETD01957 27 [ETL17]sggaaUfCfgaUfaCfuug 37usAfsaUfaCfaAfgUfaUfcGfaUfuCfcsusu uaduasusu ETD01958 28[ETL17]sgaugCfUfuUfCfuacaa 38 usAfscCfuUfuGfuAfgAfaAfgCfaUfcsusuagguasusu ETD01959 29 [ETL17]sagaaAfAfgcAfGfaacg 39usUfscAfcCfgUfuCfuGfcUfuUfuCfususu gugaasusu ETD01960 30[ETL17]saagcagAfAfdCGfguga 40 usAfscUfuUfcAfcCfgUfuCfuGfcUfususuaaguasusu ETD01961 31 [ETL17]sagugGfGfaGfAfuAfca 41usUfscCfaAfuGfuAfuCfuCfcCfaCfususu uuggaasusu ETD01962 32[ETL17]sugggAfGfauAfcAfuug 42 usGfsaUfcCfaAfuGfuAfuCfuCfcCfasusugaucasusu

TABLE 24 Relative MTRES1 mRNA Levels in Livers of Mice Dose Mean MTRES1mRNA (Normalized Group n Treatment (ug) to Group 1, Day 10) 1 3 PBS 1.002 3 ETD01597 200 0.13 3 3 ETD01954 200 1.03 4 3 ETD01955 200 0.16 5 3ETD01956 200 0.62 6 3 ETD01957 200 0.31 7 3 ETD01958 200 0.18 8 3ETD01959 200 0.53 9 3 ETD01960 200 0.69 10  3 ETD01961 200 0.33 11  3ETD01962 200 0.79

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A compound represented by Formula (I) or (II):

or or a salt thereof, wherein J is an oligonucleotide; each w isindependently selected from any value from 1 to 20; each v isindependently selected from any value from 1 to 20; m is selected fromany value from 1 to 20; z is selected from any value from 1 to 3,wherein if z is 3, Y is C if z is 2, Y is CR⁶, or if z is 1, Y isC(R⁶)₂; Q is selected from: C₃₋₁₀ carbocycle optionally substituted withone or more substituents independently selected from halogen, —CN, —NO₂,—OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷,—N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷,—S(O)R⁷, and C₁₋₄ alkyl, wherein the C₁₋₆ alkyl, is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, and —NH₂: R¹ is a linker selected from:—O—, —S—, —N(R⁷)—, —C(O)—, —C(O)N(R⁷)—, —N(R⁷)C(O)—, —N(R⁷)C(O)N(R⁷)—,—OC(O)N(R⁷)—, —N(R⁷)C(O)O—, —C(O)O—, —OC(O)—, —S(O)—, —S(O)₂—, —OS(O)₂—,—OP(O)(OR⁷)O—, —SP(O)(OR)O—, —OP(S)(OR⁷)O—, —OP(O)(SR⁷)O—,—OP(O)(OR⁷)S—, —OP(O)(O⁻)O—, —SP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—,—OP(O)(O⁻)S—, —OP(O)(OR⁷)NR⁷—, —OP(O)(N(R⁷)₂)NR⁷—, —OP(OR⁷)O—,—OP(N(R⁷)₂)O—, —OP(OR⁷)N(R⁷)—, and —OPN(R⁷)₂NR⁷—; each R² isindependently selected from: C₁₋₆ alkyl optionally substituted with oneor more substituents independently selected from halogen, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; R³ and R⁴are each independently selected from: —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; each R⁵ is independentlyselected from: —OC(O)R⁷, —OC(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)R⁷, —C(O)OR⁷, and —C(O)N(R⁷)₂; each R⁶ isindependently selected from: hydrogen; halogen, —CN, —NO₂, —OR⁷, —SR⁷,—N(R⁷)₂, —C(O)R⁷, —C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂,—OC(O)N(R⁷)₂, —N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; and C₁₋₆alkyl optionally substituted with one or more substituents independentlyselected from halogen, —CN, —NO₂, —OR⁷, —SR⁷, —N(R⁷)₂, —C(O)R⁷,—C(O)N(R⁷)₂, —N(R⁷)C(O)R⁷, —N(R⁷)C(O)N(R⁷)₂, —OC(O)N(R⁷)₂,—N(R⁷)C(O)OR⁷, —C(O)OR⁷, —OC(O)R⁷, and —S(O)R⁷; each R⁷ is independentlyselected from: hydrogen; C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₄ alkynyl, eachof which is optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S,—O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀carbocycle, and 3- to 10-membered heterocycle; and C₃₋₁₀ (carbocycle,and 3- to 10-membered heterocycle, each of which is optionallysubstituted with one or more substituents independently selected fromhalogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S, —O—C₁₋₆ alkyl, —S—C₁₋₆alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₄alkynyl, C₃₋₁₀ carbocycle, 3- to 10-membered heterocycle, and C₁₋₆haloalkyl.
 2. The compound or salt of claim 1, wherein each w, v, and mis independently selected from any value from 1 to
 5. 3. The compound orsalt of claim 1, wherein each w is 1, v is 1, and m is 1 or
 2. 4. Thecompound or salt claim 1, wherein Q is selected from C₅₋₆ carbocycleoptionally substituted with one or more substituents independentlyselected from halogen, —CN, —OH, —SH, —NO₂, and —NH₂.
 5. The compound orsalt of claim 1, wherein R is selected from —OP(O)(OR⁷)O—,—OP(S)(OR⁷)O—, —OP(O)(O⁻)O—, —OP(S)(O⁻)O—, —OP(O)(S⁻)O—, and —OP(OR⁷)O—.6. The compound or salt of claim 1, wherein R² is selected from C₁₋₃alkyl substituted with one or more substituents independently selectedfrom —OR⁷, —OC(O)R⁷, —SR⁷, and —N(R⁷)₂.
 7. The compound or salt of claim1, wherein R³ is selected from —OR⁷—SR⁷, —OC(O)R⁷, and —N(R⁷)₂.
 8. Thecompound or salt of claim 1, wherein R⁴ is selected from —OR⁷—SR⁷,—OC(O)R⁷, and —N(R⁷)₂.
 9. The compound or salt of claim 1, wherein R⁵ isselected from —OC(O)R⁷ and —N(R⁷)C(O)R⁷.
 10. The compound or salt ofclaim 1, wherein each R⁷ is independently selected from: hydrogen; andC₁₋₆alkyl optionally substituted with one or more substituentsindependently selected from halogen, —CN, —OH, —SH, —NO₂, —NH₂, ═O, ═S,—O—C₁₋₆ alkyl, —S—C₁₋₆ alkyl, —N(C₁₋₆ alkyl)₂, —NH(C₁₋₆ alkyl), C₃₋₁₀carbocycle, or 3- to 10-membered heterocycle.
 11. The compound of claim1, wherein the compound comprises:


12. The compound of claim 1, wherein the oligonucleotide (J) is attachedto R¹ at a 5′ end of the oligonucleotide.
 13. The compound of claim 12,wherein the 5′ end of the oligonucleotide comprises a phosphate.
 14. Thecompound of claim 12, wherein the 5′ end of the oligonucleotidecomprises a phosphorothioate.
 15. The compound of claim 1, wherein theoligonucleotide comprises a phosphorothioate linkage.
 16. The compoundof claim 1, wherein the oligonucleotide comprises a 2′ O-methyl or 2′fluoro modified nucleoside.
 17. The compound of claim 1, wherein theoligonucleotide comprises a small interfering RNA (siRNA).
 18. Apharmaceutical composition comprising the compound of claim 1, and apharmaceutically acceptable carrier, excipient, or diluent.
 19. Acompound comprising:

wherein J comprises an oligonucleotide.
 20. The compound of claim 19,wherein the oligonucleotide comprises an siRNA.